Unit 1 - Western Blotting

1/21/13
UNIT 1: WESTERN BLOTTING

INTRODUCTION: Wetn ltgs m t do i nfn a pcipo iia o p x iu ad iuaeuldtmn gtm l u r e h Te a e a e buts a: N b t g s rb t ia e o f d t i sei rtnn cm l mx r n s lnos e rin i o cl w i t h nm cm aot iw yD A lt e oi n h r ei g y f c e e te mt y e i s e a g. h oi n w sitecbd y . S u e adhnebcm S u e b t gR Ab t ghn a dbe N re b t gTu, w snvalt trtn ltg ol a f dsre b E M. ot r n,ec,ea e ot r lt . N lt t w s ubd ot r lt . hsi a i ib h po ib t w u r s i hn h n oi n oi e n h n oi n t e t e a e oi n d bcm Wetn ltgTe rcdrcn e rkn o nn a ee os p. eo e s rb t . h poeu a b boe dw io srs ft s e oi n e t i e 1S e ea tn f e rtn it mx rb P l c l i G ll rpoei(A E. . i spri ot po i nh iu y o ara d eE c hr sP G ) z ao h e s e te y ym e eo s 2Tas r f e ea t po i ta e bae hee in t i e te oi n . r f ot spre rtn o m m r w i ra i h r li psi . ne h a d es n l tn g e rav t o 3D t tgh po iudr vsgtn ytsei r co wta atoy n dtmntn fsi rav ts na po i oko n i. . e cn t rtn nei etao b i pci e tn i n nbd ad e ri i oi see teo t dr rtn fnw se ei e e n ii s f ai h c i e ao t z li a d es z Ipatehra uul ae ie eie t sS m w re s it oinletesrt t e rtn ea tn a stf t yItssh cs, e e o m sb n r i t e r say f n r d ts p. o e okr tnh ri gl nu h t po ispri w sas c r f iit aet m t d ute cc e e l w tm a e s a e ga o e ah e ao ia o . h e h h coe cruy eas m socm olue po is in m t d peiteh po i,n hne r l e f i t as r f e rtn ta e baeMot hsn a flbcue otfo m n sd rtn tn g e os r pa t rtn ad ec pe u eie tnf ot po i o m m r . s el y e ai h c it e e s cd f n r e h c es n eprne w re ,o ee uul o ih s p eas is say ueeal O e a tcnr t t e rtn hv spre poey nh gl tue n ot xeecd okr hw vr say mt i t bcuet uul qi ri e n w yo of h t po i ae ea t rpr o t ei o s oe f e i s , l ts e i l t lb . i ah m es ad l e s h cm e ilaaalcle m l u r e hm re , h h lwyuooo t spri ir lm wt uay tn g tlIay aem sw re d m no t o m ray vib o r o cl w i t a r w i ao o tfl h ea tnne t e iotn s in aa.n n cs, ot okr o oirh cl le od e a g ks c l lw e ao ai h ai l s t e eiec ot po itnf b s in t m m r eo tapo i Tiiiprntesrt t eeusen rntnu il ne b t fle frtn i f i y f e rtnr s r y tn gh e ba f o lrtn h sm ot t nu h t r l se a oudl nu cd yh au opo i n f n c h e a e ai e n rt e. s a o e a h st e y fe e ir es pr u rr ot glo tnfrg ai l a a f e ef m r s rn. t a e h c r a ei Background Information Ipl crmd gll t poei spri oca e m l u seusrmbtdfecsn hr adn i. rh h ca e sei m v m rr iyn n n o aya i ee cohr s ea tn fhr d o cl r l f o i r e ica e n iseMoe i l hr d pc s oe oea d ia y l e er s, ao g e e st o hf n e g z gy g e pl e cifl lgr o cl,n t s lspecl saea ra e b t gl a r t et t hs to fc m y okoe eo iopsi . h poeco e l t id a em l u sadh ee shrai hp,r e r d yh et g a rx n Teew eet a w r t t r r poi nTe rprhi er e;r c e e o s i n e td e o ee e. fs gh n t o c oa ess mwl aii t dfecsn l t poec oiy n/ gl tdtno i t bspsb r o tn fai l m c m l u s f glyt im x z h i r e ie cohrim bi ad rera ao tg eh etos l e li opr u r ar o cl. e l me e f n e er t l t o er i v e ie s uo t a c o e e Te l t poec oiy f po im l u ia t n f co op it r g op 31,seilit r g c s ti i e cipi. t p btenhi h e cohrim bi oa rtn o cl s sogu tn fH nh a e fH -0epc l nh a e l eot s l t o t a H e e t r er t l t e e e r ni e n ay e n o s oer n A c w e r pcv i e cipisto rtn w u m v iopse icosU dro-ea rg odi st nm eobnseo e f ma o p x iu opo i m y e etes l t o t w po i ol oen poi detn. nenndnti cni n, e u br fad r l dr cm l mx r frtn a s i o e r n, c es d t r i un t h o sv o e te es vrcni r lwtp . a os e b i H y d ay h Spri deo i dpns nh prse f e em t .nh cs oaa s, e oe i s p vrsne e wtt aa s cnettnTe oe i o ea tn u tse eed o t oe i ot gl ai It ae fgr et prse i l ae i r l i h gr e ocnao. h prse f ao z e z h r x e o h z m y i v sy h e o ri z pl c l i gl w i a pl ezd sh gl f m dle i dc ae a t cnettn fem nm repesd s ) c ae;o ee is l aet b o ara d e, h h r o m re a t ei o e,kws er ss sh ocnao ogl oo e( r e a %T i r sshw vrt ao fc d y y ym e s c e y i e sr i e e e ri x s ne ,i s f e t et tfrs lkg.nr sgh cnettn frs lkre teoh tam nm repesd s )po % b w i ter ssh prseA oe %C h x noc si aeI e i t ocnao oc si erav tt o l oo e( r e a %C u t5 y e hdc ae t oe i. bv 5 , e e on c an e ri on li e t x s g e e z t prsenr ss gibcueh c s lkr iezs i ie tf mm repne gl h oe i i e e aa eas t r si ed re wt t loo oe xadd e. e z ca n e on m i h sf r s Motrtn a fcoa d n o ara i r g grm5 t2%T cn in 25 . e lg po i,uh sm uol un,raos rtn gr a sa spo i r r tnt o pl c l d a i f es e a i e y y m e n n o %T o 0 ,otn g -%C V r a e rtn sc a im ng blso vru po iag gt ,r ai yr es o i i e e e e r o e o aa s gl S a pl ul ter m n ( t1 b a spre o pl c l i gl lgrulc c ssc a r t tnr m n ,n grs. e l d n gr e e. m l o nc od fg et u o k)r ea t n o ara d e; r nc iai ,uh se r i fg et o aa e sv o s l y ei a sp e ad y ym e sa e e d si o a c s o F r pm l sli ,lpc s f tethu b ra e b t gl unte o p ty xl e. e t seag ot sm lcm oetit wd tb svd o ot ar o tna sei oie ssol ee r d yh e btob cm le ec ddWhnh i r e f e a p o pnn so i o e i e i e uo l e n r d td e , el u ez n h e s o e e b a eoa i lprsea r i tem y e sd y glf sg oe i, g d ngl a b ue. ne z ae Denaturing Vs. Nondenaturing Gels E cohr iopo i m y e a i otneehr ea rg ro-ea rg odi sN ndnti bfra ,for ,eue w eeeb l i lcvy ut l t poes frtn a b cre uudriednti o nndnti cni n. o-ea rg ue r ocu er id hnvr io c ati m s er s es rd t un un t o un fs e s qr o g a it b ra e,log dnti bfrcn e sdf e ia a tr a rb l i lcvy frl t poei Adnti gl h h lw dt tn fny ac cvy ee i dahuh ea rg ue a b ue ihr s w yo e te io c ati aee cohr s ea rg ew i ao s e co oezm t ati tn t un fs t e n u o g a it t e r s. un c l ei i it fl i r oaot dntat cld n cvy e N ndnti bfra ao sd hn hr dfecs rko no xt h h a g e r t r o tnhn oo n e vlf e ea r i ae a ati gl o-ea rg ue r l ue w e ca e i r e a nw tei w i m y i g a re li t lw g m h u ns l it . un fs e s g f n e e s c v e e s uo a spri o t bs oseA ea p w u b t r o tn f oy e. ea tn nh ai fi. n xm l ol eh e li oi zm s ao e s z e d e s uo s U dro-ea rg odi st p m sb cruy e c do aii ca e i r csIiue l ko t i e cipis f e rtn oie siodro nenndnti cni n, e H ute a flsl t tm x z hr dfe e.t sf t nw h s l t o tot po i f tet ret un t o h el ee me g f n e s uo e oer n c h es n r n cos t bsp . hoeh etH e Icnatfn t m l u ri ireath po i a dnte wtS d mddclltS S(hpo16; br O br 16)S Sia ai idtgn n ots i l h o cl sese vn t rtn r ea r i oi oeyua D Sai,97Wee& son 99. D s n n n e r t r , oy e e a z l , e es e ud h u sfe r , oc e e w i dnte po i b wap g rudh pl ete akoeIs di , D cn ra engte hr tt pl eten rpr notl g . e ta d h h ea r rtn y r i a n t o ppd bcbn.n o o gS S ofs nteav ca eoh o ppd ipoot ti e t Whnr t c u s es pn o e y i n e i g e y i i o s nh ee wtS Sad r ui aeth pl ete bcm rd ongte hr s i eul hr dni so ca e euieg . i eh ca e euieg ot S S i D n ae c g gn t o ppds eo eos feav ca e wt qa" a e esi" rhr prn l t S c t hr prn l t f e D h d n ,e y i i g h c g t e g tnh n e g tnh h pl ete o p xs os n t e ci l r eee o t cm l prn l g iao os n Tu t mg tn e cyso l dtmnd yh sv g fcot o ppd cm l icnt th l t af c xrd nh o p x euie t s l cnt t hsh iao vl i itay e ri b t i i eetf e y i e a, e er o e t c e e tnh s a. e ri o t tl e e e en f h ara i m t s t trtn a spre acri tt i o cl w i t c l d ai oh po i r ea t cod goh r l u r e h ym e r x a es e ad n e m e a g. At uh otrtn bhv nrayn D gll t poei ism ot t r oneht eehi es n m i aoeadht t asm tn udri t log m spo i eae om l iS S ee cohr s t iprnt e gi t t t n u ia e pi ln,n t ih s pos ney gh h es l er s, i a o c z ah c q r c af e u i ln e m t d rntadt r us inteeal Tu f ea p , po iwtaa e xes fc ir i e m y e b des D t n t i lrtnad ec m v e o a ovl, ee l wl ob ri e hso xm l a rtn i lg ecsoaiced s a w l i l S S h ay c po i n hne oe h e i h st l lb . r e e h r d su ln s a p a e, anrayn D gll t poei bom l iS S ee cohr s l er s. Te a rsfnsotsyt itdtmn t m l u r e h oiprmx r opl ete. h idn b r n g gl i s na po i oko n o cl h m j ue l s f iss m so e ri h o cl w i t f u iu s fo ppdsTis oe yu i a ewt t dr rtn fnw m l u r o ue h e e e e e a g s m e te y i s nn h a d es e a w i ta n wtt pl eteo e hr tidAlere tnh eis e enh l 1 ot m l u r e hoa o ppd adtR. y esrg fht , e e h l g i h o ppd tb ca c re. i araosi xtbt e t o 0 f e o cl w i tf pl ete n i fB m aun R, ai t gs o he y i a ez n li p s w eg h e a g y i s i t sh d t c f mt sm lw loh pl eted i d yh d t c f mt sm lw loh dern a t dr cr cn e ee t iw i t l ot m l u r ia er h a p e tt o ppd,id b t ia er h a p e tt y f t s na u e a b gnre n h hh o f e o cl sn o e e l e y i ve e sn o e e l e o, a d v ad c eg h e a w i tt drss lt aa st i f h m l u r e hoa uko n rtn a t n e e ri d y esrgtR icm asnoh s na sFredtlo e hs na ip td git e R. e o cl w i tfn nnw po icnh b dtmn b m aun i f o pro tt t dr . uhr e i n g a d oe n hr T e a g e e e e i s n i ea d t as btdnti ad o-ea rg ess m a poi dnh Appendix tProtocol 1.3. o ea rg n nndnti glyt s r rv e it h un un e e d e o Teoo i t lg e a ee lu en f t ara i cnettn eddo i e D po i odfetis h fl n a e i s gnr gi leo h c l d ocnao nee tsv S S rtn fi r se. lw g b v a d i r e ym e ri e es f n z e MW R neo ag f r %TS SPo i D rtn es 1% 1, 04, 0 5 2 0-5 0 0 0
www.med.unc.edu/pmbb/PMBB web site/mbt/WESTERN.htm
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1% 1, 07, 0 0 6 0-0 0 0 0 5 6, 020 0 % 0 0-0, 0 0 0 Molecular Weight Markers Te a a u br fi r to m ray vib po im l u r e hm re w i a ue tet a t m l u r e hopo i o Wetn lsTe gnrl hr r nm eodfe cm e ilaaal rtn o cl w i t a r h h r sdo sm t h o cl w i tfrtn n s rb t hy ee l e e f n e cl le e e a g ks c e i ee e a g es e o. ay fl tto a grspetnd n us i dPetnd r yd a e ehrae lh po i it m l u r e hl drtnd i a i lde rlr te ec a iow ct oi,r a e ad ntn . r a e o de m r r iehv a t rtn nh o cl w i t des i wt sg y o ae avl ah ln e e si ae si kst l e es e e a g a ae h ne tn i y ot po i it l dr s i d i a i r to r deTe i r t s i d a e , heh m sepni ,aeh avn g opoin ua b uu f e rtn nh a ei tn wt dfe cle y. h dfe l tn m r r w i t otxesehv t dat e frv i nm i os h es e d s ae h f n o d e f ny a e e ks l e v e a dg g i nfao ot sei po i it l drTipeetcn s n so h h o cl w i t a ebn yu rl k g t h h a sm t e ocr hn o e f e d tctn f e pci rtn nh a e h r n of i a tw i m l u r e hm r r ad o a o i aw i cn o em s cuw e sm ot ei i i h f c e s e d . s v s uo c e a g k e on c i h sg de m rebnsu o t gl yd a e , e f ehv to a avn gsTe ao t spri ot po i o t gl b r dyoo e drg i l yd a r ad r fh e D e m r r t ro ,aew m i dat e. hy lw h ea tn f e rtn nh et ee i fl d un ny k n fe . k sh e r n a l e ao h es e o a l lw i e cohr iadhyni thweiet po i w rtnfr f mt gl t hbiztn e baeD e m re ,o ee ao ae ee lidat e. h l t poes n t i c e o f i l rtn e r s r dr h et h yri i m m r . yd a r hw vr l hv svr d avn gsTe er s e da f ny e s e a e e o e o e c d ao n ks ,s a s a adi ode tt m l u r e hm re de aet oe l oiy f ee rtn,oht e cu c om l u r e h dtmndrmt s m re m y ob di n fysoh o cl w i t a r os lrh vrl bi ot s po i s t t acr y f o cl w i t e ri f h e a r a nte t o e e a g ks t e am l t h es ah a e a gs e e o e ks a pei a dse. di nl, e i r trtn o cl iay i n ad o ob dh sm a onode h hed ta raei ot po ipasTe s r s s eidA di ayt dfe po im l u sn n g e bn d nti t a e m utfy w i l so bodn g f e rtn ek. h ce r t lh f n o e e e e v n e c a n h e s bi ot depo icm l cn l b m rnl ec,o r m re , bid tlsolnte oe m rt n n mntad hu b l dd nh gl ii t iy f e y-rtn o p x a ao e a i . necle a r i oe aa,hu ob bid oeh oe i e n sol eo e o t ewtn al t h e e s ga H o d k sf l l d l a u d a e h a huaeduo f mt s c tpeet socl it bns n orfr itnr h t ko r nl s fo r h ad. t li o e o v o on e U s i d a e a r wtt po ism l oie sadhn iazd frr s roh hbiztn e bae y tn g i a rtn tnTee a e hv t ntn m r r r u i h rtn a p s f tetn t vul aetnf tt yri i m m r b s in wt po is i hs m r r aeh ae ks e n h e e e nr e s i t a e e e d ao n ai h e a. ks e avn g opoin sa bns hs m bi hs obe ae d yh adi ode. hs s na shroe i t bset a s f o cl w i t hy aeh dat e frv i hr ad w oe oiy a nten lr b t di n fysTee t dr t e rg eh etsm t om l u r e h Te hv t a dg p l t te e t o a d ef v e i e e a g. e pia d avn g it t e d ntlwoeo oirh por s f e eo tass sh tnf eiec. o ea udh rav d avn gs f ew m j r r idat enh t y o oao n tm no t rg sot glro s s t r s rf i yT gtr n t e te idat e ot to a r m ys a ah l t e e h ee e a e f n c o e li s a h o t e om l u r e hm re , ay ep r btpetnd n us i d o cl w i t a e o t sm gl y s f o cl w i t a r m n pol u o r a e ad ntn m l u r e hm r r nh a e e p e a g ks e n h si ae e a g ks e . Separation Of DNA Or RNA P l c l i glcn l b ue tspre m l N fg eto< 1bTe ea tn fob -r ddi anc iaifg etiet lbsd n i,i eh o ara d e a ao e sdo ea ts a D A r m n f = k. h spri odulsa e lerulc c r m n s nry ae o sesc t y ym e s s a l a s ao e tn n e da s i e z n e ca e esysh sm f p cs fi r t nt Te a esr f sg -r dd ulc c fg etw e dnte b ui h h ocnaos fr it glr hr dni it a eo i e odfe l g . h sm iteo i lsa e nc iai r m n hn ea r y sg i cnettn ou anh eo g t e re f ne h e u r n e tn e da s ud n g ri e e b o em asC n raoadfecs a ao e sdo eo e o nc ods nesei ccm t csCoe ccl D A f ea p , ra e tdfet y t r en. of m tnli r e cn l b ue tr l pl ul te udrpc liu s ne. l d iu r N , rxm l i e r do i r h o i f n e s sv y e i a r a s ra o e s td f n e et tdpni o t dg e fue oi ,n t cm l ety tns f dp x N fg eta sm t e b spre o a o-ea rg eaesm l x n eed g nh er ospr igadh o p m n rsad oa ul D A r m ncn o em s e ea t n nndnti glfra p es n e e cl n e e a r e a i ad un t e dntao de r u altdfecsn eodrsute ea ri u pe m b o i r e iscna tc r u tn s y f n e y r u. Two-Dimensional Electrophoresis Itod es nll t poei po i a fcoa ditnh bs ooe hsapoeyadia eod t ,nh bs oao e Moto m n , ol t nw -i ni ae cohr s rtn r r tnt f o t ai fn pyi lrpr,n, scn s po t ai fnt r scm ol i e ci m o er s, e s e a i e r s e s c t n e e s h. ys er c f ui iue f t f ties ncnei t cre ot aue e ad D e cohr iia l gl ue f t scn d es nTe ot i lue poeu o sgs sdo h i d ni ,ovn ny a i ui t gl n S S l t poesn sb ei sdo h eod i ni . h m swd y sd rcdr c n r er m o s e l rd n b , er s a s re m o e e it tf ' r l7. s ay s 10 ii trtn hv be r o e f ma i lsm losr b ts e o. sh oOF rl )A m n a 10 d tcpo i ae ene l dr sg a p fe m yh m t d a ae( sn es sv o ne e u i h Continuous and Discontinuous Buffer Systems Acnnos uess mioen h hh sm bfr ue togot e ead to e coe. otuu bfryt seue n a i ll eogl hy r otuu bfryt s n iw i t a e uei sdh uhut gln abt l t dsC nnos uess m r i ol sg a r fe Te a i f e c e f s r h h er i f e q r y ne y . e s p tstpad roe aeutf sc proe a m noi a ezm prctn ro pea te l t poeioa ailpre po i Te a ao sdo i l o eu,n a fn dqa o uh u ss s oirg n ny e ufao o f r ri e cohr s f pr l ufd rtn hy r l ue f me e t e r p tn i i i r p av e r s ty i a i e. es r nc iaie cohr i Te a d avn g it t e a p m sb ia ocna dom sc ec r o e bn wl e s i a t dp ot oinla p i ulc c l t poes h m i idat esh t sm l uten cnett f ,i e ahe l d ad ib a wd sh et f e ri sm l n e d er s. ns a ah e re r n sv l e e h h ga e t wl h e. e l Icnat d cnnos uess mcnett ec sm lcm oet ta a o bn ko n sh "ak. h oinla p m yhroe e uh oe it ad n ots a iotuu bfryt ocna s ah a p o pnnio nr w ad nw a t s c"Te ri sm l a t e rb m c m rdu ,n r, s i f e re e n r et ga e ef le cm oetw i bn c s t e ecn eeo e m res . o pnn h h ad l eo t ra b r l d oe ai s c o gh sv l y Ad cnnosyt ue bfrodfeto psi ad Htc a a iotuu vlg ad Hg d n Te i r tuezns rs bid y t at o iotuu ss m ss ue fi r cm oi n n p o r t d cnnos oae n p r i t h dfe bfroe a t ie b al st s i e fs f n e t o ee s i t ae. f n f e e al z e w dfetel e . i r gl yr f n a s e Te pel e tog w i t sm lps sits nw a t "ak g e.t aa eprgl h hs o- sii tt po ism l Te ueiw i t h upr yr h uh h hh a p as f , ko n sh s ci glIi lg-oe e w i innr t teoh rtn a p . h bfr h hh a ,r c e e e r i s et n " s r , c er v c e e e f n c e s ci gl m d cn i a i ( ul a ai ) hs e cohrim bi ig a rhnhtf e rtnw i t t k ue o e coe ue m scn ia i t k g ei ae otn no u ay n n nw oe l t poec oiys r t t t ot po i heh a bfr rl t d bfr utotn no a n s as n s l o er t l t ee a a h e , l e n f, e r f, a n w oe oiyseshnhtf e rtnA e cohr ibg st "ai i " t s ci gl oe f t t nh po iadevs ei i zn ol e hs m bi il t t ot po i s l t poes ei, el d go i h t k g em vsa e h t rtn n l e bh dt oe f w r l t s a a h e. e r s n h e n nn e a n sr a e e a n a o cnuti. h h hroae r i tf izn cue t po itm v f t ado s c"t e ona btenh l d g n tigos odcvyTe i evlg g d notsoe assh rtno oea e n t"ak at budr e e t e i adrl i . it g t ae h e e sr t h y w e an ai n n B l t s ci gl a epr yr fewta m lr oe i,nw a t r o i o spri gl h gl pea dn bfr fi ecnettn n p ( a e w h t k g ei deel eogl i s aeprseko n sh e ln rea tg e Ti ei r r ia ueoh hrocnao ad H i n o ea n s a h l z e svg an . s s p e f g ri n ai iss m.nh evom n t m bi ot tigo i r ssohts ona m vs ha ot po i Te rtnseo e ionidabns cod go n n yt )Its nin eth oiy f erl i n e e s t i budr oe aed f e rtn h po iir l dn i v ulad acri t oc e i r ,e l t h ai n c a n at y h e. e sv t d i n seadnh cs oa o-ea rg e acri tsae i;n it ae f nndnti gl cod go hp. z e un , n The Laemmli System Te ot i lue ss mf e cohr iopo i ipoalt tecbd y am l5.t a iotuu ss mf r o i po i dnte wtS S Te h m swd y sd yt o l t poes frtn s rbb h dsre b Le mi )Ii d cnnosyt o e ln rtn ea r i D . h e e r er s es ya i ( s s i e r svg es ud h l d go it Le miuess micli ,n t tigo ig c eA cri l t r o i gln t s ci glrm d u iTiH lue (fi r t e i i nh am l fryt s h readh rl i s l i . cod g , ee ln eadh t k g ea ae pn r- C bfr odfe an n e bf e od e ai n y n n ny h svg ea n e s fs f n e cnettn n p )w i t t k ueiTig c eAlue cn i0 % S S ocnao ad H, heh a bfr r-l i . l fr otn . D . ri l e n f s s yn b fs a 1 Membranes Nt cloe n rn r d V Fm m r e a gnrlue iWetn ltgF r r r o cl w i trtn btt e ofe w rs e. o ee i or rtn i e l ad e f c P D e ba s r ee l sdn s rb t . o lg m l u r e hpo i o y s flr ok w lH w vrf upo i r us o io e n e ay e oi n ae e a g es h p is t l ,y e ilshn 0 b i cloe hu b ue bcueta a uh i eb d g aai. h P D m m r e wl ow r w l net s ccm t cs se t 1 k nr eus sol e sd eas i s m c h hr i i cpcyTe V F e ba s int ok e udrh e iu s ne. s a t l o d h g nn t n l l e r a
General References
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1A dr nN ad GA dr n17. w -ies nll t poeiohm n lm po i. rcN tA a. c U A7: 2-45 . ne o, L n N ne o.97T od ni ae cohr s fu a p s a rtn Po. a. cdSi S 4 4152. s s m o er s a es l . 5 2C hnSea 18. xm lonndnti glwtcnnos uess mf prctn f po itb asyd y f i b d gJBo C e .5: 2. oe, tl 92Ea p fo-ea rg e i otuu bfryt o ufao oa rtno e s e b ait i i .. i. hm 27 55 . e un s h i f e r i i i e a f y nn n l 1 13. 51 3D v, J 94D srtn fiotuu bfryt f nndnti glie cohr i A nN . cdSi 0: 331 . ai B 16. ecpo od cnnos uess m o o-ea rg e n l t poes n. Y A a. c 29 7-8. s ii s i f e r un s e r s. . 3 4Jv , M 17. o pt-evd e co obfrei so m l hs zn e cohr i A nN A a. c 29 7-9. .oi T 93C m u r re sl tn fuer p f ui ai oe l t poes n. Y cdSi 0: 746 n ed i ei f ce r t c p er s. . 4 5Le mi K17. l vg osutapo i drgh asm lot ha obc rpae 4N te 2: 0 . am lU 90Ce ae ftc r rtn un t s b f e ed fati hg T . a r27 8. , a r u l es i e e y h eo u 6 6Ma uiaP ad RB r s 18. D mc sbi ag d npl c l i gll t poei A a Bohm 8: 636 . t d r T n D u es 97S S iol lerr i to ara d ee cohr s nl i e .7 8-9. s a, g . ra n ae y ym e er s. . c 3 7OFr lP 17. i r o tnw -ies nll t poeiopo i JBo C e .5: 0-01 . ' rl H 95Hg e li tod ni ae cohr s frtn . i. hm 20 0742. a e, h s uo m o er s es l 4 8O ntnL16. i e cohr i-: akrud n t oyA nN A a. c 11 2-4. . r e , 94Ds l t poes I cg n adh r n. Y cdSi 2: 139 si c er s B o e . . 3 9R ie , Aea 16. c ibfryt f r o tn faoipo i. a r15 8. . efdR tl 92A ic uess m o e li octn rtn N te 9: 1 sl . d f e r s uo i c es u 2 1. t k n, W ad Pe.92Le mie;sdo spri og cpo ihroe.. i. hm 27 9426. 0Sr l dT n D ut18. am l l ue f ea tn fl ortn om nsJBo C e .5: 5-90 ia c t gs r ao y e l 2 1. o b , ea 17. l t poecr s r frtn f mS Sad c /e gltnrcll ePo. a. cdSi S .6 3045. 1T w i H tl 99E cohritnf opo i r D n aiu a e o i eu s. rcN tA a. c U A 7: 5-34 n . er t a e es o d r s t lo o l . 4 1. brKad O br 16. ais d om l u r e hdtmntn y D e cohr iia otuu bfryt .. i. hm 24 4641. 2Wee , n J son 99B s t y f o cl w i te ri i b S S l t poesn cnnos uess m JBo C e .2: 0-42 . cu e a g e ao er s i f e l 4
PROTOCOL 1.1: ISOLATION OF PROTEIN FOR WESTERN BLOTTING AND PROTEIN QUANTITATION
INTRODUCTION: Po i cn e ai pea do Wetn ltg y uie yl ofeehwn,oi tno liwtezm s n dtgn . m aa cla uul qi es t rtn a b es r r f es l p e r s rb t b m l lcc s f ez t i sn ao,r s i ny e ad e r t Ma m ln es r say ue ayo y e oi n t p e r a g ci ys h e es i l e l t le n gnrld nteuer z t wn o t ezm t ta ett trncs rtle esad ea bc rlesWhe n cnye a m ln es icyn y ad ee l o or i feeh i rh ny acr t n h a ees yoy yatn cri ati cl s ay qr e a g e i em s a e a s tn ea l i oe a l m m aa cldet i . l s i l r l t sm lbfrsdo S SP G e cohr i ts uecn i bo ohnll , D ad e at t nl h h tuietocnao cnn rrwtt h a p ueue f D /A E l t poes h bfrotn rm peob eS S n m r p e aow i asf i cnettn a iee i h e e f r er s, i f as u c oh c f n c ri tfe h e cm olue Baf d rtn s yH net bstleh clia oi d a p bfr h h otn l eog l e ot s ieen sei tao acre o m n sd r o po ias . ec is et y t esn m di sm l uew i cn i o nuhe l f een rrg pc so lw cu t y dr e a i os e l f e e f c as w vs h tfi e l a dtmntn f e rtn ocnao.n ucs w f n t t e lo in bo ohnll peet ayn rr c wtt po ide i i asyO c t cl e ri i ot po icnettnIorae eo dh m ry mt g rm peob e r n d n iee ne i h rtn y b d g s . neh es e ao h e ri u a e t i u v e tfe he e nn a e l a le, e solb bid r y dt y hu e oe immediately ia c wcpueo ea ray rt ssht a r a aten a p bfr es h d l n sr a t tdnte n po ae t m ye i cv ism l ue e b u e a mn i e f. Iodro sm thwm c po itepciora p s n solcni rh fl i . o m m aa cloe b i aot g ew i tfesrmair f n retet a o uh rtno xet usm l oe hu os et oo n F r a m ln es n otn bu1 w t e hoclf i e e n e d d e lw g i l as g lo l o t e m d . s mn t clhv g w taot0 clm ts i s 0 clw t e hprr . i e clsogl9% w t ad er ee tn ees rtnw cn ei A s i h es ae r no bu16 es l ig e 19 es ew i teg m S c a e iruh 0 a rn nay vrh g l ipo i e a a u ge l o l h v / l g a n l y e l yi s e, vrruh et a t t0 clcn i0 g frtnS i 0 m ocla r upne i1 lfa p bfrh cnettn ib ruh 1 m /l vl e f e ogl sm t h 19 es otn . opo i of 0 lfes r e seddn m osm l uet ocnao wl eogl 0 g . o m o y y i ea l a 1 e. 1 l e s e f e ri l y mA u aot0 cnhn eae f dtmntn f e xcpo icnettn bu1 mla t b t no e ri i ot eatrtn ocnao. e k r e ao h e ri PROCEDURE: 1Sa big a r a . . ttoi w t bt r l n e h 2S idw t clia oi letueueIm d tye oeh blot spr t tn vrxo eupn t pltPoed s u k a psb . p o nh esn cn acnig t .m eie r v t u f e ue a nad ot tr sedh ee. rce a qi l s os l n e l c r f b al m e k h na e s e l. cy ie tog t nxs p iodroncvtpo ae t ta r iy er eh po isoie s h uhh ett sn reti ta rt ssh cna d dg d t rtn ) f tet r e e ai e e a pl a e e( n r . 3A d vl e fomt pru 2 Sm lB frht eul t vl e f eeupne cl ee . d a o m oro e e te X a p uet i qat h o m ot r sedd e plt u m ar e f as o e u h s l l. 4V rxo eupn t pltn le est nr s resm eie ta l n . ot tr sedh eeady cl h tnf clim d tyo c a screw cap mc f eue sg i t a. a ad rce ts p e s e l s l e a e l , al e io g t ui P em nC p n poedo t 5 ru b n p t e immediately. 5P c t sr cp io g o l ko mc f euen oi w t f 25 i, e ciinc utyu rr d tuet h ta d a p cn e . l eh c w a mc f e r c t io g t ibig a ro - mn t n hl ii nlo a e yo s iTi r t sm l a b a e e ru o p ru b l n e r .h lt e i e a . s ee e s r f zno fu r s te r e f u r u . odo r te n 6Te oe etct d tb vrvcu deoh peec oh h o cl w i th m sm l N .f e xat t vcu tp ee ai,oi t . h bid xat nso e e ios u tt r ne fi m l u r e hcr oo aD A It etci o ioso i t es sn a l r e ys e s g e a g o h r sos pt l y ce t sm lwtto hrpls f 5 eod ec ui a iorb sttbu5% flo e It sm lm ks s ayps"on drg ah u th h a p i w sotue o~ scns ah sg mc poe eaaot0 u pw r f e a p ae a t d "s tsud un ec br, e e e h s n r l . h e e s i st D Awl e ha d N ib ser . l e Atnte , a oi t intvib ,ash etcbc adotsvr t e tog a 9 ag hpdrinelui a m srg bf eod g n lr i l i sn a r oaaal pst xatak n f h ee lm sh uh 1 gue yoe c ed sg 1 lyne e rl i a e av y f co s le e r r ai r m e n i o an aqoot sm lo t S SP G glf e a p isl ios frh 1 gue han,hne ed s n plh sm lbc adottog a 5 lutf e a p nh D A E e It sm l st vcu aet 9 ag sergcag nel ad u t a p ak n f hh uh 2 i h e e . h e i s l t e i e le e r r gue ed . h sn ao m t dsat ad ai t n sg srg ad ee l l d tc aespri s nh gl ag nel Te oi tn e o if e n esrh ui a yne n gnrl e so l nrea tn o t e. e ci h sr e a n i ay a e ao e s 7S it sm lf 3 scn mc f e1K ,e oeh spr t t n p c in c a mc f eue . p h a p o 0 e ia io g (4 )r v t ue a n ad l et a l n io g t . ne e r ru m e na, a i e ru b N t Whn a p s rsn a dh s p a nte eesrbcueh cla s t ruh d r t t to eeif m d fretui t sm l o : e sm l a oi t tst m y ob ncs y eas t es r oh ogl iu e h n plt o e aecnig gh a p . e e e ce i e a e l e o y sp d a lsr t r n e f e Te f ei o cniet osr n pltfrh si o ih s p hro , yu ost l be e o eeaet p ,mt i t . er f s ny v l t e n ts e 8D tmn t po icnettn f e utnt tProtocol 1.2) . e ri h rtn ocnao ot sp r a ( e ee e ri h e an . N t Te m utfrtn eddo ec l e eed o t sni i ot s in m t do e sdad l o t d t uo opo ia og ea t o :h a onopo inee f aha dpns nh esit f e tn g e o tb ue,n ao nh ir tn frtn m n spre e e r n e t y h ai v h s e si i b e a bnsWi C o as Bu,o cn e ca lla 1 ia i lbn; i t 10fd oe esi s es i,bu1 n. h ce imnset ad. t om se l yu a dt tsie s mgn sg adwt h 0- lm rsni e i rtn aot0 gTe hml i cn h i e e t t ne he o t l as v v u e t hi e ue tdt tngn po ia f m rsni ehnhs tapo is i. e n us sdo e catei rtn r a oe esi t t eo lrtn tn c q e i c e er t a e t v e as
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MATERIALS:
1. 0.5 M Tris-Cl pH 6.8 C m o n A o n/ m A o n/ 0 l o pud m ut0 l m ut0 m 5 1 TiC 3 g .g r-l. 6 s 0 0 D i id 2 4 m 9 m e n e H O 5 l5 l oz A j to H6 wt 1 H l d st p . i M C. u 8 h A d e n e H Ot 5 m t 10 l d di i d 2 o 0 l 0 m oz o Al te o t n o o l ro t pr ue e rm k g h f aaj tett te H T iibcueh p o Tisl i s rt pr ue eedn ad er s apoia l0 3 H lw h sl i t cot o m e e trbf e ai tei ld s nso h p . h s eas te H f r o t n a e e tr dpn etn dc ae p rx ty . p o uo o m a o n n um s s uo e m a e m e 0 frah o i r s it pr ueF rxm l a . M sl i hs Hvl s f. 8 ,n 8 a5C 2o ,n 37o C r pcvl o ec 1C n e en e e tr o ea p , 0 5 o t n a p a e o 9 ,. ad . to ,5C ad ca m a . e 0 uo u 5 9 5 e et e . s i y Sele y uol i . t i b atc v g rz i an 2. 10% Sodium Dodecyl Sulfate Sodium Salt (SDS, FW=288.4, also called Sodium Lauryl Sulfate) C m o n A o n/ 0 l m u t 00 l o p u d m u t 0 m A o n/ 0 m 5 1 D i i d 2 40 l0 m e n e H O 5 m 90 l oz S S 0 10 D 5g 0g Heat to 6o t as t i o t n 8C o si d sl i . s s uo A j t Ht 7 b ad g f do s fo cnr e H l d sp o . y d i ae rp o cnet t C. u 2 n w ad A d e n e H Ot 10 m. d di i d 2 o 00 l oz Dses iaq osT e in ne t s rz 1% S S i nen lu t hr s o ed o t i e 0 D . p i . e el i C ui : aa a when weighing SDS and wipe down the weighing area and balance after use because the fine crystals of SDS disperse easily. at nWer m sk o 3. 2X Sample buffer (Minus Bromophenol Blue) F aC net t n tc S l i A o n/ m A o n/ m i l o cnr i Sok o t n m u t 0 l m u t 0 l n ao uo 1 5 0 2 M TiC p 6 0 M TiC p 6 2 m 1.m . 5 r-lH . . 1 s 8 5 r-lH . . l2 l s 85 5 4 S S 0 S S .m 2 m % D 1% D 4 l0 l 0 1% Gye l.m 1 m 0 l r 2 l0 l co 0 1% 2 r potao1 m 5 l 0 - c te n l. l m Me a h 0 H O0 m 3 m 2 . l. l 5 5 Dv en lu tad r z. id iaq os n f ee i i e 4. Boiling water bath. 5. Sonicator or 1 ml syringe with 19 and 25 gauge hypodermic needles. 6. Appropriate centrifuge tubes and screw cap microfuge tubes.
PROTOCOL 1.2: BRADFORD PROTEIN ASSAY (BIO-RAD REAGENTS)

REFERENCES: Ausubel et al. 10.1.1; BIO-RAD Protein Assay Kit Instructions; and Bradford, M.M. 1976. Anal. Biochem. 72:248-254 INTRODUCTION: Tivrs p po cl r e rin po icnettn a lglr l e t er r i lue L wy s yTe s y eed o t f t aw e cride ( r h e i l rt of dtmn g rtn ocnao hsa e e a dh ai wd y sd o ras . h as dpns nh a t t hn ea ysh e s ym e o o e i e ri r y pc e l e e a a e ch tn e, C o as Bi nBu R20 b do rtn t y ne o siit i bobneB m aun t taasrac at si d ae ntoe a t sedy sm t om se ri t l -5) i tpo i h udr a ht h r srac. y esrgh o lbobne t e ht w vl g n cnh r i et a i l l a e n es e g fn e a i e t h f e e h u al i e t cnettn f e rtn o tn i r pctsm s na po i N tt toa po i g e ndnclbobne ht oht eehi ele ayehi e h ocnao ot po isli wt e et o e t dr rtn o h ntl rtn i a i taasrac si s t t t n u,k m n t n us e ri h e uo h s o a d e. e a l es v ei f , ah c q i c q ue tm au po icnettnir l olue l ro prge te ocnaos f e a e rtnIr l , e m utfrtn e ri drmt s na sdo esr rtn ocnao, e l n sf f cm an rav cnettn ot sm po i ne i t a onopo idtmn f h t dr e e ri s ay y u o i li ri h e . at h y e e e o ea d cr m y a f mt asle rtn ocnao b aat otoo o m r u e a vr r h bo tpo icnettn y f o f fd r oe v yo e u e ri cr w l . O e f e oto m n ue w y tdtmn po icnettns i t de om se ri t l R20G nrl t s na cr s sdo e ri po i n ot m scm ol sd aso e ri rtn ocnao iwt h y C o as Bi nBu -5. ee l, e t dr u e ue tdtmn rtn h y e e e ri he i l l a e ay h a d v e e e cnettn ui ts e o cvr r g f m0o 0 mg fieloy e r S prt dr 5 l atnA a , iiprnf yuo ne t dht e ocnaos sgh m t d oeaa er ri n i h n o t10 oehr szm o B A es na m r co. gi ism ot t ro tudra t t t y a d e i nt a o sn a h C o as Bi nBu R20 tn i s ea po i m rr dyhn t r Tu, e sm topo icnettnn e la s ae wtt pr u r rtn e g om se ri t l -5 s ib d cri rtn oee i t o e . hst et a frtn ocnao icly t vrs i h ai l po ibi i l l a e a n tn e s al a h s h i e e ri lse i h e t a c e n ue f t s na cr . S adyoy e rgnrlcoe bcuehy ri xese n r dy b i b ih h ut H w vrB A ipr u rt d t sdo h t dr u eB A n l zm a ee l hsn eas t a n pni ad e i otn l n i pr . o ee S , ai l, nso rea d v s e ay e ee v a l aa e g i y , n t a e c dnte ai ad os og e pr u r acre sm tot po icnettnn r e e la sIiiprntntt te teo a r r E t libfr ea res n de nti a ai ll cu tet a f e rtn ocnao ic d cly t .t m ot t o h rav tw t o T , eys ue u l y v t ay c a i e h e ri u lse s a o e a li e h s f hs sni nasrac b ie. ec t s na cr ,hu cn it sm faLs B frocnao a t sm l tpeetr nos sm t opo i a a i i atbobne yt lH neh t dr u esol otnh a ei lyi uecnettn sh a p so r ne oeu et a s frtn gf c sf ea d v d a e n s f ri e e v r i e e cnettn ocnao. ri N t Teehi lhet to e wtt Baf d s yis nm eocm o bfro pnn ,uh s D , aiee wtt asy bv a ea cnettn o :h t n aset acm s i h r o as l a u br fo m n uecm oet sc a S S t t tfe i h s aoe cri ocnao. e c c h h e dr a s t f s h n rr h e a tn ri A o ecm olue asy sgh de inhn ai(b i b f mt P r C e i l o pn) m rr iat S Siee nePr p t bsw yo vi nt ro m n sd s ui t y b i oi c otn l r h ic hmc C m ayi oees nt D n rr c. e ash et a tao h y a n e cc c d aa e o e e e a s st o tfe h e d psb dtgniee nenhs asy ito i e r n f mt libfr(rs dtgn sc a Tin h h fnn rrls O e a t n s a oi t o os l e r t tfec it e s sso mt tg t r h ys ue o ue e r t uh s r w i oe iee e ) n cnh ue sn a r r ie e e n rr e a d e e s o e s fs e es t o c t tfe s . e co hm gnetpea t etc . o eeits aet iprntpoi aeutpo aenit snh libfro r eter ao ot sm luti a b o oeiro r r h xat H w vr h cs ism ot t rv e dqa rt s i bo it ys uetpe ndg dtn f e a p nl cn e z pe e r s ni i a o d e e h ir e s f v a i h e it asyd n bidn a p libfr s e ad oe ism l ys ue a l e s f.
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PROCEDURE: 1Dleh de i f ro m s f . w t adi r uaye a i pr u t .Tis p ib dn b t T s . itt y wt o vl e oDI a rn feotn r in ai le (h t wl e oe yh A ) u e h u u . e l t m ng t a s s e l c e . 2R m v eog po ietctg e r snb cl it BoR d rtn s yt i l 15 )n du do 0 ml i w t ia lset b. . e oe nuh rtn xat i ae oal o r h i a Po ias (p ay - ml d it t10 wt a r g st t e e r ov a e on e e a y cl a le h e n a su 3A d m odu d y mxoh du d rtn xat i It mx rim d tyu s a b e ttvrn uees xat ovr li o a ual . d 5 lfit de itt it po ietc mx f e iu m eie tn dr l s roead s l etc C ne e f u r nb le e le e r , . h te al r k u a s r . sy y e e tdt ty y ay i r c bten s y n b n ad oe xat o e cb ee n dfe e e e as ad l k d m retc e f n e w a a r . 4MesraO 55 maa sb n wtbfrn de . au t D 9 n gitl k i uead y. e n a h f N t Te r f d egnb d thyo lsTee en t ts o r d oscte a p sh cle de ud u o t cvt w lad r etad o :h Ba o r eti si t tg s hs m ash a yue cneuv sm l t o r y bis p nh uee as n pe n n e dr a n gl a. a a i e e od l e t l v s acreed g f e a p O . hsfo a ui g scvtst y uteie ot i e aobten a p so e oeh bud y. n lr te cu tr i ot sm l D Tu i u r sg ls uee, e m sb rsd uwt t nle e sm l tr v t on deA ae av a an h e y e n a t h n hh w e m e tn i apoc itue i oalp sc uee w i a ravli xese prahso s d psb lt cvts h h r e te n pni . s e ai t c e li y e v 5B cm asn i a t dr cr (r a d y A )et a t Ymg m utfrtnnh Xmlde tt s na 5 l atnTe cne t . y o pro wt s na u epe r b T s sm t h i h a d v pe , i ee a onopo iit e e addoh t dr m r co. hn ovr h ea d e i te cnettn sm t( mg mlo / o mg l ocnao et a Y / ) mg r / . ri i e X t ml m
MATERIALS:
1. Bio-Rad Protein Assay Reagent (Proprietary Solution Of Coomassie Brilliant Blue R-250) 2. Spectrophotometer and disposable cuvettes. 3. Protein Standards from 0 to 100 mg per 5 ml reaction Y u h u n tf mte i us nn h i rd co t tipoootate a p b f ro tn a u br fo p u d tacnne e wt te sa- prcl S SIiip r n n t o so l oer h d csi iten o ut n o h rtcl th sm l uf cna s n m eo cm o n shta i r r i h asyi a i a D . sm ot t o d o s o t i s h e e i tfe h n tu r t a t ece te lw d o cnr i s fhs i e e i sbt csn h asyF r D te aiu cnet t n o pt lwt te sa i0 % ia 0 usm l (o a o p tlt f o xed h ao e cnet t n o teen r rn u s ne ite sa.o S S h m x m o cnr i cm a b i h asys . n 10 la p . r cm lei o l ao tfrg a m ao ae h 1 eF e s i e e i sbt cs e te i a m n a In addition, as in any assay it is important that the standard curve contain exactly the same components in the same concentrations as the samplesn r rn u s ne seh Bo d aul tfrg a R ). except of course for the substance one is measuring. Wi tee o s e t n imn ,e iaalfro s ut g s n a creo te i a poe asyPea B Ao L sz e tc sl i s to cnr i s f m / l w t . t hs cni r i sn i hr s t eo cnt cn a t dr uv frh Bo d rtn sa.r r S r yom s k o t n acnet t n o 2 g m i a r h d ao d e b r i a d r i pe o uo ao n e N t te t drs l o tn f acnet t n f.% S S10 sm lw i ite aiu a o n o sm lb f r5 l o pt lwt te sa. oe h s n a a cna ai lo cnr i o 0 , a d l i n ao 1 D / mla p h hsh m x m m u t fa p uf (u cm a b i h asy 0 e c m e e ) ae h
2 gm s c ( )XSm lb f r< 5 ) 2 ( ) i l g o poe bi add o m asy m /lt kml o 1 a p uf (= ml O ml n m s f rtn e g d e t 5 lsa e e H Fa i n 0 90 55 5 91 500 1500 5 83 2500 5 75 5550 0 4 10 T e ad ah 0 mltc sl i so spr eu e o tn g m o f e d rtn egn ad ed 5 5 ae5 i hn d ec 10 s k o t n t a ea ttb cna i 5 lfi r poe r etn r A 9 . fr mn o uo a in le t i a a t .
PROTOCOL 1.3: POURING AND RUNNING SDS POLYACRYLAMIDE GELS (SDS-PAGE GELS)
SEPARATION OF PROTEINS ON THE BASIS OF MOLECULAR WEIGHT: SDS GEL ELECTROPHORESIS References: B. D. Hames and D. Rickwood Gel Electrophoresis of Proteins, A Practical Approach, David Garfin in Meth. Enzymol. (1990) 182:425-441., Sambrook et al. Molecular Cloning 18:47-18.59. SOURCE: HOEFER SCIENTIFIC INSTRUMENTS INTRODUCTION: Te a toye obfryt sn l t poei cnnos n d cnnosAcnnosyt hs n a i lspri gln ue t sm bfr t t k ad hr r w t s fuess m ie cohr s otuu ad iotuu. otuu ss m a ol sg ea tg ead ssh a e uei h a s n e e p f e er s, i s i i e y ne an e f nen t gln d cnnosyt , m t ditee pd y r tn16)n D v (94,n lepplid y am l17)a or t tea e oe e cld h e Ia iotuu ss m a e o f dvl e b O n e (94 ad ai 16)ada r ou re b Le mi 90, nn sii lg prgl ae a e . s i e h r s o si s t az ( er v r c , l s ci glsae d no oa ea tg e(s re e ta t r o i glEc gl yr m d wta i r tue adh t k ue a dfet o t gl t k g e il r o t f spri gl l e r do sh e ln e. ah el ei ae i dfe bfr n t a bfr r i r f m h e a n , ye p an ao fr e svg ) a s h f n f, e e n fs e f n r e e bfr Teeo tn b i b ia iotuu ss mim c g a rhnhtf cnnos n f r sn tb dsre. o ee t s p rotuu ss mia ue . h r li otn l n d cnnosyt s uh r t t t oa otuu oeo e oso e ecbdH w vr h i l cnnosyt s fs s uo a a e s i e ee a a i ra i ,e m e i e llesro eu. ie ai tstp t t e Ia iotuu ss m a rtn m bi, qat te esrot mg tna oa hr d pc sn n l t afl iie eie e enh m bi ot bfr n d cnnosyt , po is oiya unti m au f e iao re f ca e sei ia e ci le , n r d tbt e t oiy f e ue s i e e' l t iv a e h ri t g e e r id s tm a c w e l t h f i it s ci gled go)n t m bi ot bfr nnh uprak tigo)Whn l t poeiis rdt i s n t po i s rtmg tioh o nh t k g e(ai i adh oiy f e uei it pet (ai i . e e cohr sstt , eo adh rtn tt ia n t n ea n l n n e l t h f o e n rl n n er s ae h n e e s a o re t e s ci gl h po i cnettia e tn oe ae t s c,e enh l d go adh tigo. h po i cnneo ia it s c utt yec t t k g e Te rtn ocna n vr h zn cldh t kbt e t e i i n t rl i Te rtn otu tmg t nh t k nl e r hh a n . es re yi l ea w e an n e ai n n es i re e a ih a e spri gl h cnettn frtn de ntcuia otuu ss mw e t po i et t spri gl a oe er a bod sh oinla p . s ea tg e Ti ocnao opo i os oocr cnnosyt hr h rtn n rh ea tg ei zn nay s ra a t ri sm l A a an . s ri es n i e e e es e e an n l e ga e r u, cnnosyt poue bnshtrtc ad or r o e. e la otuu ss m rdcs ad t a h k n pol e l d st i e a ei y sv A adi aavn g oa iotuu ss mit t eeer ecn ei a yt tw r wta ai l po i F rxm l h cn s bfrw i df ol n di nldat e f d cnnosyt sh t r a hra dsn ss m o ok i pr u r rtn o ea p ,e a ue ue h h i r n t o a s i e ah s c g e h t a c e. e fs c f e y s hynhi oii s t t po ioie swlt k he t r rtn o cn mnn wl o l t it r bi soh a rtn f tet is c w i o epo i rot i t int i l e m le g t a e nr la l h es a as l . Te rcdrw g e a be aat ta igl ra Whea egl ra g e l r r o tn n hv aa ecpcyt yaeogro u ado m n h poeu e i hs en dp do mn ef m t i lgref m t i c a re li ad ae lgraai, e t l etr n f ay e v e i o . l r o s v e e s uo r t h k n n r
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proe t i r sd eo tnhy rv es neesr u ssh n e e r li t poi iuncs y p e ca s uo e d a.
PROCEDURE: Te E20 gtS a Iia ia rvr asb euin ne f r ie cohr iopo io nc iaism l os a vl eT o x c glad i e o h S 5 Mi y m lI mn te ei ll gln ieddo a d l t poes frtn rulc c a p s fm l o m . w 7 8 m esnwc s f h l s iu t a c tt r p er s e e d e l u h ehr o ara i o aa s glm y eu s uaeulo t S 20P l c l i gla r ui t g sad l i mp t poi d i t uiTe oe f iepl c l d rgr e e a b r i lnos nh E 5. o ara d e r u sgh ls n a mn le rv e wt h n. h cro t y ym e o s nmt y e y ym e s e n n e a u u as d he t t ui e e a a etxhnetog w i co nm y e iu t . h n sr s s haecagrh uh h h ol t a b ccle e t v r c a r ad Preparing A Stacking Gel Te aul acm s i yum dll t poeiui idsre o tpuglui t tpcieu m n Te aipoeu ,o ee wl eh sm im s h m nat to e wt or oee cohr s n wl ecb hw o ore sgh sei qi et h bs rcdr hw vr ib t a en ot h h er s t l i s n a f c p . c e , l e it csF sdpni o t pr u rpa t bi ue,o m sasm lehrw g sp t ,r sg g sp tad o hd l i p t spre b to n a e. iteed g nh ai l apru e g sdyu uts b ieto ls le o a i l ls le n nt e a mn le ea t yw sn r n e t a c as n e et a as ne a a c u a a, ad p sc pcrw i dtmn t tcnsot gl neh p t ad pcra asm l , e oo hso e el wtt e rgrs o c m e io csn r lt sae h h e ri h h kes f e e O c t le n sae r s b dt btm a tb sa d i a o aa e rl pdn a atgi ai s c e eei h . e as se e e h t e hp o a t i g w i peett gls m lf ml k gTe yu orh r o i glpo pee ri d ett t dpnetnh dp osm lit w lse i r bl ) h h r n h eas b r e i . hn o put e ln eu ta r tmn dp h i eedno t et fa p nh e ( d g m e w. c v se e y o an e svg de e h as e h e e le a a o Te ee l lit t e iac btenh btmot w ladh t ot r o i gl 2o 4ie t dp osm l it w l Tenr s id t c bten h gnr resh t d t e e e t oo f e es n t o f ee ln ei t- t sh et fa p snh es h i e en ia e e e au a h sn w e t h l e p h svg s m e h e e l . ca sn w t btmot w l n t t ot r o i gl i w l etot sm ls p rl tt f t aiae m rd t c f t po i tb f ue io bn w e h oo f e e adh o f ee ln ewt e dp f e a p i l ee sh a t t t s oe ia eo h rtn o eo sdn a ad hn e t h l e p h svg h l h h e m y fc e c h t k sn r e es c t t sm lisr d uoeaa ed t c it w l h a p spe otvr lgr ia enh e. e e a r sn e l
Teeo i glhu b pu d u k aet T ME ad m oi pr ltiaddoh ara i gl iu . h bspoeu itwt r t ara i mx h r ln esol e or qi l frh E D n a m n m e ua s de tt c l d emx r Te etrcdr so ida h c l d i svg d e cy t e u sfe e ym e te e h w e ym e ia aportse srg o d psb p ee n tao t gl itfwdw oe i ot asm l p t tao tpi bbl. neh r o i gl n n prpie id yne r i oal i t ado lw h emxol o n n s e f e s b d le o vi r p g ub sO c t e ln ei a z i s e pt l e o d h e e as da n e e svg s pu dthu b cvr wtt R ni G l vry ueo i bt otec d oye w i i itpl eztn f e c l i at t ot gl t r a or i ol e oe d i h un g eO ea bfr r ou nl xl e xgn h hn bs o m rao ot ara d t eo f e e Wida e s d e he n l f s a o u c hi y i i h ym e h p h . h w s a a onot ara i sli io Ps up ee r l e o en mc f eu ad a thy oh por s f e o m rao cn eedyoo e.f e m l m utf e c l d o tnn a at r i t o p c sm ia io g t n cpi t s t rg sot pl eztn a b r i fl dIt l h y m e uo t e pt a ru b gl e e h y ii a l lw h ara i it p ee rueaso a e, e t ria rb mwtt ara i mxAl id u br fei so m k gh s ci ad eo i glrg e it c l d nh i t o t flthr nt nh es pol i h c l d i i t nm eor p f ai t t k g n r ln ea i nnh ym e e p t b i d h e e h e ym e . m e ce r n e a n svg ev e m tisetn a rlsco. ea i N t May o pn se a atgr t tlw yuo orrm1o ara i glaaie o : n cm ai sl csn tyh ao s o tpuf e e l i a a l o t5 c l d e t t . ym e s m At t r o i gla pl ezd i. hnh pl c l i it Ps up ee a hree)puo t i bt oo bfro tn n asm lt s ci gl frh e ln ehs o m re ( w e t o ara d nh at r i t hs a nd,or fh s u nlr uesli ad s b h t k g e e e svg y i . e e y ym e e e p t d fe o a f uo e eea n ara i gl i O c cm lepuit s ci gl iutiece t t ot p t . h cm it n e aa ag ad n cr r srd erh upr f r c l d emx ne o p t or h t k g emx nl r hsh o f e le Te o bsh hl tn nl n oe on iee nat pelt ym e . e, nea n it a e p h as e d e en t e eo rhhn s e fe O c oe onr f e o bsnee,et l et opse onr f e o b e g a f nt tp ub s sh cm iiee ioh t o i tad i ogl ne n cr ot cm ii rdgnyo rh poi cr ot cm bi cru ot r bbl a t o bsn rdn t o f g d . e h st l w e t e h n el o a e e st t e p t gl d adi as ci gl ii o e a be l tun t cm iei . oeay xoe gl iu wtR ni G l vry ueo i bt oad lwt h e A d di nlt k g emxf m hs eno drgh o bn r nC vrn epsd emx r i un g eO ea bfr r ou nln ao h e . t o a n s s i e st o te h n l f s a l e s ci gl pl ez. l eh r a i gl iu ia at r i t o mc f eueonu t to m rao ipoed g rpr. t k g et o m reP c t e in emx r n Ps uP ee r io g t ti r h pl eztns rcei poey a n o y i a e m ng te e pt ru b se a y i i n l At t s ci glo m re adh cm hs en et r oe, e eir d tueTe e cn e r pdn lt wa ad l e a4Cad sdh nxdyf frh t k g epl ezs n t o b a be gnye vdt gl e yo s. h gl a b wa e ip sc r n p cd to n ue t eta i e ea n y i e l m h sa s p ai p a e ncs r Te ignrlolmn al s feo i aiyh nxdyH w vris or o m ne t t e e b s r l grhn n dy shy eit ees y hr s ee l n im l sor ln bi t eta. o ee t nte m eddh t gl e t e o et oe a a t bg o a. e ay y i o svg l e t ,i c ah s o d n a e n dtia a t y rotn df i ocrbtenh s ci ad eo i gl e rre sh dy uad i s n cu e e t t k g n r ln e eo t e f o u s w ea n svg . Sample Loading and Final Assembly of the SE 250 1Ca ph snwc aa st crwtt a mn p taa st gse ad rn d i t nt u. s t r c m so l ph gl p c. . l t ad i git e oe i h l i le git e akt n oi t wt h o h pU eh e l p tc m t ei l e m e h n h heu aa n h , ee h e c e da a e na 2Ma t l ao ot w lui a a epn n t n et r oeh cm . . r h o tn f e es sg m r r e adh gnye v t o b k e ci h l n k e l m e 3Rneh sm lw lwtt k uead rn ynei t ui vr sk . i t a p es i a bfrn da b i r gh n oea i . s e e l hn f i vt e t n n 4Fl e a p w ladh uprn l ebfrhm e wtbfrEc upr ueca bro s bu7 m. h cpcy f eo ebfrhm ei . it sm l es n t peado r ueca br i ue ah pebfrhm ehl aot5 l e aai ot l r ueca br lh e l e w f s h f. f d T t h w f s apoia l10 l prx ty 5 m. me 5A d / vl e fXbo ohnll sli tt po ism l ad i Tihl btivuln t sm ldrgod g n idtmn g o . d 1 0 o m o1 rm peob e o tnoh rtn a p s n mx h e s o n iazgh a p un l i adn e rin hw 1 u u uo e e e . s p h s i i e e i an e i eelad aiyh po i a bi spre o t gl n o etci desc a peor wl ok s e. vn n r d t rtn r e g ea t nh e A y t rr k g y,uh s hnl d iw r a w l y p l e es e n ad e . h a n e l l 6Icon idse,onct t i f mt i ecrta o w t f ctrei ri uiad eitccle ol t eo m ne co n f ue .folgs eidcnet eu n r h n roeo cl a ra eo rre tn n,n bg o iu tco n R cm edd ol t o s i r h bg o e n d e u f ao t g n ra a. as r wtts olg oe rw t o 5% w t ad 0 e y n g cl o oue nfee rn a oobsd iu a t s wlr pr ldm g t ui i h con cra a r r 0 a rn 5% t l e l o D nts atez o ay l hlae mx r sh e iiea b a aeh n. hi i e e e he y . i r c te e lr a y e t
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7L a t glsgl tpd euni tsTei solcr t a yu s o l dB crutdpesh P em n l la yuodh w lo s o . odh eui ftpe sqec gi . h tshu u eo r o a yuo . e a f o er t i t a s wy s o l t e s a nt e n ai n p p d v w d a el s e pt o a e l tb wyusm lotf e e. l o m som d y i r to b a g e iTb I e w o l ora p uot w lWe vl e f e b dfe cm s r i nn al bl : o e h l l u r f n e ev e o TABLE I VOLUME OF SAMPLE PER 1 MM DEPTH Ti nsoC m h kes f o b c N m eoWes u br f l l 5w l o b - e cm l 1- e cm 0w l o b l 1- e cm 5w l o b l 0 m . m 5 6 ml . 4 2 ml . 4 1 ml . 5 05 m . m 7 9 ml . 5 3 ml . 6 2 ml . 2 1 m . m 0 1.ml 2 7 4 ml . 8 2 ml . 9 1 m . m 5 1.ml 9 1 7 ml . 2 4 ml . 4
8P c t sfyio t ui n aahh l d tt pw rup . . l eh a tl nh n ad tc t e soh o espl a e e d e t t ea e y Running the SE 250 1G lm y eu aehros ncr no cnt toaeC nt tu et tdi ay sd i a iotuu bfryt s t t ea o . e a b r tiecnt tu etros nvlg. os ncr ni r i nl ue wt d cnnos uess m oh t re f s n t a r a t a r s at l o h s i f e ah t e cohrimg tn ir a cnt t ruhut r . sg Le miess ma4 m (0 Apre cnt tu et i to . m tc gl l t poec iao wle i os ntogot eu U i a am l lyt t0 A 2 m egl os ncr n wt w 0 5 m h k e, er t ri l m n a h h n n g e ) a r , h 7 i s t r solb cm lenutvrn orTe or pni vlg ad aae ei shu b apoia l10 ( rieoe rw gl ad h u hu e o p tij oea hu h cr sod g oae n w tg st g sol e prx ty 6V f ehr n o to e)n 4 e n d e s . e n t t t n d me o t s w t/ lr w t/gl F ri er o tnao ecr nst g a b aval N vxeo m nsun ghi r aTiGyi glt cnt toae as eo 8 as e. o h hre li , l ru etei m y e dib . oe r m ed r i t r e t r/l n eaa os nvlg tg t2 s g s uo w r t n s e c nn e p c s ce a t o15 f 1 r gl f2 V o o 2 e. r s 2Whnh tci deece t btmot gl e wt src ot l ebfr tn fh pw rup ,ionct l d,n r oeh l ot . e t r k g y r hsh oo f e e( l h uae f eo r ue , ro t o espl d cnet ee sad e v t i f e ea n a e t h bo e f h w f) u f e y s h a m ed h ui n. t 3So t fwoco ntog t i ecrad ionct t i . e oeh i ecrf mt l ebfrhm ead or ut upr ueb . t h l fol t ruhh n roe n d cnet eu n R m v t n roer h o r ueca brn puot e pebfr y p eo a h en s h bg en o ew f h f i ei t croea i . n r gh oe vr sk vt e n n 4R m v t s e l p adiec glad i o t cr . e oeh i c m s n l ah esnwc fh oe ed a f t h fe . 5U e lt gl egso roe t glad i F O T EB T O tao bek gh erot nt e a mn p t Te ewl say tkoh . s p sc ew de tpy pnh esnwc R M H O T M o vi r i t a f e o hd l i le h gl iuul sc tt ai e h d an e s h c u a a. l l i e a mn p t R m v t sae ad el e eo t p tio ty ftn ri te l i le e oeh pcr n pet glfh len ar os io fav. u a a. e s h fe a t a a xi
Care and Maintenance 1Rneh l ebfrhm eadnecrwtd te w t aeec ue . i t o r ueca brn i roe i iid a rfrah s. s ew f n h sl l e t 2Gas n a mn p t solb c ae ia t n dtgnsc a B -5t nie t ruh wtt ad iid a rB tp t cn l b ta d . l ad l i le hu e l ndn sog e r tuh R S 3, e rsdh ogl i a n d te w t. o le a ao er t s u a as d e r ee h n o y hp sl l e h as s ee wtcrmc c c ai sli . i h iai l n g o tn h o d e n uo 3S ae solb w se wtdu d B -5t nie wtt ad iid a r . pcr hu e ahd i it R S3, e rsd i a n d te w t. s d h le h n hp sl l e 4D ntu c v o bih ui rn oi p sc as . o oat l e r o ts n o ay fs lt pr. oa li t t ai t STOCK SOLUTIONS FOR LYSIS BUFFER AND RUNNING AND CASTING SDS PAGE GEL N t U eh fe aeom oTiad l i w e m k ghs sli sTee "r- l sdnhs f m li ss nni tnht e Hot sli iaj t o : s t r bs f s f r n g c e hn ai t e o tn. h tm TiC" e it eo u tn ia i c i t t p f e o tns d s d e ee r s yn n e uo r s u e r ao d ao a h h uo ue wtH l i C. h
1. 2X Sample buffer (Minus Bromophenol Blue) F aC net t n tc S l i A o n/ m A o n/ m i l o cnr i Sok o t n m u t 0 l m u t 0 l n ao uo 1 5 0 2 M TiC p 6 0 M TiC p 6 2 1. . 5 r-lH . . 1 s 8 5 r-lH . . 2 s 85 5 4 S S 0 S S .2 % D 1% D 4 0 0 1% Gye l.m 1 0 l r 2 l0 co 0 1% 2 r potao1 m 5 0 - c te n l. l Me a h 0 H Ot 1.m t 5 m 0 o 2 o0 l 0 l Dv en lu tad r z. id iaq os n f ee i i e 2. 1 X Bromophenol Blue C m o n A o n/m A o n/m o pud m ut l m ut l 1 5 Bo o hn l l 1 g m rm p eoBu m 5 g e H Ot 1 l 5 l o 2 o mt m 3. 10% SDS
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C m o n A o n/ 0 l m u t 0 m o p u d m u t 0 m A o n/ 0 l 1 5 S S 0 5g D 1g 0 H Ot 10 l 50 l 2 o 0mt 0m o
4. 0.5 M Tris-Cl pH 6.8 (FW=121.1) C m o n A o n/ m A o n/ 0 l o pud m ut0 l m ut0 m 5 1 Tibs 3 g .g r ae . 6 s 0 0 D i id 2 4 m 9 m e n e H O 5 l5 l oz A j to H6 wt 1 H l d st p . i M C. u 8 h A d e n e H Ot 5 m t 10 l d di i d 2 o 0 l 0 m oz o Al tee pr ueo o l ro t pr ue e rm k g h f aaj tett te H T iibcueh p o Tisl i s rt pr ue eedn ad er s apoia l0 3 lw h t e trt cot o m e e trbf e ai tei ld s nso h p . h s eas te H f r o t n a e e tr dpn etn dc ae p rx ty . o m a o m a o n n um s s uo e m a e m e 0 p frah o i r s it pr ueF rxm l a . M sl i hs Hvl s f. 8 ,n 8 a5C 2o ,n 37o C r pcvl H o ec 1C n e en e e tr o ea p , 0 5 o t n a p a e o 9 ,. ad . to ,5C ad ca m a . e 0 uo u 5 9 5 e et e . s i y Sele y uol i . t i b atc v g rz i an 5. 0.5 M Tris, 8.8 (FW=121.1) C m o n A o n/ m A o n/ 0 l o pud m ut0 l m ut0 m 5 1 Tibs 3 g .g r ae . 6 s 0 0 D i id 2 4 m 9 m e n e H O 5 l5 l oz A j to eid Hb ad g o cnr e H l d st dse p y d i cnet t C. u r n ad Al tee pr ueo o l ro t pr ue e rm k g h f aaj tett te H T iibcueh p o Tisl i s rt pr ue eedn ad er s apoia l0 3 lw h t e trt cot o m e e trbf e ai tei ld s nso h p . h s eas te H f r o t n a e e tr dpn etn dc ae p rx ty . o m a o m a o n n um s s uo e m a e m e 0 p frah o i r s it pr ueF rxm l a . M sl i hs Hvl s f. 8 ,n 8 a5C 2o ,n 37o C r pcvl H o ec 1C n e en e e tr o ea p , 0 5 o t n a p a e o 9 ,. ad . to ,5C ad ca m a . e 0 uo u 5 9 5 e et e . s i y 6. Initiator - 10% ammonium persulfate (FW=228.19) C m o n A o n/0 l o p u d m u t1 m (H ) 2 31 N 42 O g S D i id 2 tvl e e ne HO o o m oz u T e o t n a b s r free l ek a4C h sl i m y e t e o svr w es to . uo od a 7. Running Gel Overlay (0.375 M Tris-Cl pH 8.8, 0.1% SDS, used to prevent oxygen from reaching gel while polymerizing) Final Concentration Stock Solution Amount/100 ml Amount/500 ml 0.375 M Tris-Cl pH 8.8 0.5 M Tris-Cl pH 8.8 75 ml 375 ml 0.1% SDS 10 % SDS 1.0 5 ml H2O to 100 ml to 500 ml
8. Tank Buffer (0.025M Tris pH 8.3, 0.192M glycine, 1% SDS) Final Concentration Stock Solution Amount/1 L Amount/4 L 0.025 M Tris base pH 8.3 3.0 g 12 g
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0.192 M Glycine 14.4 g 57.6 g 1 % SDS 10% SDS 10 ml 40 ml H2O H2O to 1 L H2O to 4 L
Because of the pH of this solution need not be checked, it can be made up directly in large reagent bottles marked at 4.0 liters. 12-16 liters can be made up at a time. Reuse the buffer in the lower buffer chamber 4 or 5 times, but discard the upper buffer after each run. 9. TEMED (use required amount straight out of the bottle). 10. Casting the SDS PAGE Gel The following recipes for SDS PAGE stacking and separating gels are for a 40% premade stock of 29:1 acrylaminde:bisacrylamide.
A. Preparation Of Separating Gel

In a 100 ml sidearm flask, mix 40% acrylamide solution (29:1) with water, Tris HCl (pH 8.8) and SDS, according to the chart below. Optional. Degas under vacuum to 10 to 15 min. to remove oxygen which inhibits the polymerization. Add 10% ammonium persulfate and TEMED according to the chart below. Swirl gently to mix. Use immediately. Draw up some of the excess mix into a disposable Pasteur pipette or place about 1 ml in a microfuge tube and cap tightly. If the gel fails to harden in the pipette or microfuge tube, there is usually a problem with the persulfate, TEMED or both. ML Of Each Solution Needed To Make ~25 MLs Of SDS PAGE Resolving Gel With The Following Final % Acrylamide Concentrations Stock Solutions 5 6 7 8 10 12 13 15 H2 O 15.17 14.55 13.93 13.3 12.05 10.8 10.17 8.92 40% Stock 29% acrylamide 3.13 3.75 4.37 5.0 6.25 7.5 8.13 9.38 1% Bisacrylamide 0.5 M Tris HCl 6.3 6.3 6.3 6.3 6.3 6.3 6.3 6.3 pH 8.8 10% SDS 250 ml 250 ml 250 ml 250 ml 250 ml 250 ml 250 ml 250 ml Ammonium 125 ml 125 ml 125 ml 125 ml 125 ml 125 ml 125 ml 125 ml persulfate TEMED 25 ml 25 ml 25 ml 25 ml 25 ml 25 ml 25 ml 25 ml There are a number of venders, such as Bio-Rad, that sell premixed acrylamide solutions at defined acrylamide:bis acrylamide ratios (e.g. 29:1). It is cost effective to buy these solutions and more healthy, too, because you, or your employee, does not have to breath acrylamide dust, a neurotoxin, during weighing.
B. Preparation Of Stacking Gel

In a 100 ml sidearm flask, mix 40% acrylamide solution (29 acrylamide:1.0 bisacrylamide) with water, Tris HCl (pH 6.8) and SDS, according to the chart below. Optional. Degas under vacuum to 10 to 15 min. to remove oxygen which inhibits the polymerization. Add 10% ammonium persulfate and TEMED according to the chart below. Swirl gently to mix. Use immediately. Draw up some of the excess mix into a disposable Pasteur pipette or place about 1 ml in a microfuge tube and cap tightly. If the gel fails to harden in the pipette or microfuge tube, there is usually a problem with the persulfate, TEMED or both. ML Needed To Make ~25 MLs Of SDS PAGE Resolving Gel With The Following Final % Acrylamide Concentrations Stock Solutions 4% H2 O 15.8 40% Stock 29% acrylamide: 1% Bisacrylamide 2.5 0.5 M Tris HCl, pH 6.8 6.3 10% SDS 250 ml Ammonium persulfate 125 ml TEMED 25 ml
PROTOCOL 1.4: PROTEIN TRANSFER FROM ACRYLAMIDE GEL TO MEMBRANE

INTRODUCTION: Protein blotting is succinctly described by Therese Timmons and Bonnie Dunbar in Meth. Enzymol. (1990) 182:679-692. A description of the process can also be found in Sambrook et al. on pages 18.60-18.75, and in Ausubel et al. 10.8.1. There are two major methods used to transfer proteins electrophoretically to a membrane, semidry blotting and tank transfer blotting. Tank transfer blotting wastes large volumes of buffers and is not very versatile since only a single buffer can be added to the tank. While many workers continue to use it, it seems to possess no advantage over semi-dry blotting. Transfer by diffusion blotting, which is
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analogous to the original procedure developed by Southern for DNA, is possible. However, because of the highly cross-linked nature of the acrylamide gels used for protein separations it is extremely slow (days). Semidry Blotting Reference: M. Lauriere, Anal. Biochem. 212: 206-211 (1993) Semidry blotting is becoming increasingly popular as it is fast, uses little buffer, and the progress of the transfer can be readily checked. A wide variety of buffers have been used. The procedure we will adopt is more complicated than most since it uses 4 different buffers. However, it has the advantage that both high and low molecular weight proteins can be quantitatively transferred. This is because the buffers are cleverly designed to efficiently elute the proteins out of the gel and deposit them in high yield on the membrane (see the diagram below). Buffer I, closest to the cathode, contains a high concentration of SDS at an alkaline pH. This buffer serves as a continuous source of SDS during electrophoresis and helps to solubilize proteins. The SDS-protein complexes will be negatively charged at the alkaline pH and readily move to the anode. Buffer II, which is just underneath buffer I, reproduces the electrophoretic conditions of the SDS-PAGE separation with respect to pH, ionic strength, and presence of SDS. Under these conditions the resulting SDS protein complexes in the gel continue to move rapidly to the anode. However, as the proteins approach the membrane these conditions change rapidly. Buffer III contains no SDS and has a low pH and a high methanol concentration. This buffer is designed to stop most of the proteins at the surface of the membrane. The acidification of the medium promotes the dissociation of the SDS from the proteins. Once the SDS is removed, most proteins become less soluble and lose their negative charge that stops them from migrating farther toward the anode. The high concentration of methanol in this buffer also tends to favor protein precipitation at the membrane. Buffer IV raises the pH of the transferred proteins to 10.4 at which pH basic arginine and lysine residues are positively charged. Thus, any protein that fails to stop at the pH 3.8 boundary, will acquire a net positive charge and be directed back towards the cathode giving it a second chance to bind at the membrane. While this four buffer system may represent biochemical "overkill", it may be useful for blotting very valuable samples or high molecular weight proteins which can be difficult to transfer quantitatively by standard methods. THROUGHOUT THE PROCEDURE WEAR GLOVES TO AVOID GETTING OILY PRINTS ON THE MEMBRANES WHICH CAN INHIBIT TRANSFER. ALSO BE SURE TO AVOID BUBBLES AND EXCESS LIQUID WITHIN THE GEL SANDWICH. TREAT THE BLOTTING APPARATUS GENTLY AS THE GRAPHITE ELECTRODES ARE SOFT AND EASILY BROKEN. 1. The gels and blotting membranes sandwiches are assembled with Whatman No. 3 paper and blotting pads in blotting sandwiches as shown in the figure given below. The composition of the buffers is as given in Table I. 2. Dry blotting pads large enough to completely surround the gel to be blotted are wetted with the desired buffer. The Whatman paper is wetted with the desired buffer and the membrane filter with deaerated water. (If using PVDF membranes, they must first be wetted in methanol, then water.) 3. Equilibrate the gels for 15 minutes in Buffer II. (The gels will swell slightly). 4. Assemble the gel sandwich as shown using a frame of polyester transparency film (from overhead projectors) around each gel to prevent buffer mixing and electric current leakage. 5. Transfer for 1 h/mm of gel thickness at a constant current of 1 mA/cm2. Note: If you are transferring many gels, you can place a wet sheet of dialysis membrane on top of the bottom stack, then place a second stack on top of the first. The dialysis membrane will serve to ensure that no proteins from the upper sandwich migrate through to the lower sandwich. 6. After removing the blotted Immobilon-P filter you must first dry it to fix the proteins to the membrane before continuing on to any transillumination or rapid immunodetection procedures to ensure optimum results. There are four drying options. a) Soak the membrane in 100% methanol for 10 sec to drive out the water. Then place the blot on a piece of filter paper. Wait for the methanol to evaporate (~15 Min) b) Place the blot in a vacuum chamber (secure the blot between two sheets of filter paper (~30 min.) c) Incubate the blot at 37oC (~1 hr) d) Place the blot on a lab bench to let it dry at room temperature (~2 hr)
MATERIALS
1. 120 mM lactic acid (FW=90.08) Compound Amount/500 ml Amount/1000 ml dl-Lactic acid syrup (85% W/W, Sigma L-1250) 5.26 ml 10.52 ml 1.209 g/ml = 11.4 M H2 O to volume 2. 200 mM Tris base (FW=121.1) Compound Amount/100 ml Amount/1000 ml
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Deionized H2 O 70 ml 700 ml. Tris base 2.422 g 24.22 g
Allow the solution to cool to room temperature before making the final pH adjustments. H2 O to volume and sterilize by autoclaving. NOTE: If the solution has a yellow color, discard it and obtain better-quality Tris Also, make sure the pH electrode being used measures Tris accurately. Many electrodes will not measure Tris accurately. 3. Transfer Buffers I through IV can be made as follows: Buffer preparation from stock solutions (for 100 ml final volume) Buffer Stock A Stock B SDS Methanol Final composition of Solution (ml) (ml) (g) (ml) buffers I 50 50 0.4 -- 0.4% (w/v) SDS, 60 mM lactic acid, 100 mM Tris, pH 8.4 II 12.5 12.5 0.1 -- 0.1% (w/v) SDS , 15 mM lactic acid, 25 mM Tris, pH 8.4 III 50 10 -- 20 20% v/v methanol, 60 mM lactic acid, 20 mM Tris, pH 3.8 IV -- 50 -- 20 20% (v/v) methanol, 100 mM Tris, pH 10.4 Note: Stock A, 120 mM lactic acid; Stock B, 200 mM Tris base.
Tank Blotting This is a commonly used alternative to semidry blotting. Tank transfer uses much more buffer than the semidry procedure. The following protocol is written for a large tank transfer apparatus (Hoefer) used for an overnight transfer. NOVEX makes a small combination apparatus that allows you to do both the Western and the subsequent membrane transfer with the same device. If you do not already have a small acrylamide gel box and a semi-dry blotter or a tank blotter, then this is a good buy because you get both pieces of equipment at a substantial net savings. NOVEX also sells very precise precast gels for about $8.00 each.
The Hoefer procedure is as follows. 1. Cut two sheets of Whatman #1 paper and one sheet of nitrocellulose to 8 cm x 5 cm. Wear gloves. Do not touch the paper or nitrocellulose with your hands. Fingerprints can show up on blots (that way you know who has been touching the nitrocellulose). 2. Carefully wet the nitrocellulose in transfer buffer. Start from one corner and slowly lay the filter in to the transfer buffer. 3. Soak the Whatman paper and nitrocellulose in transfer buffer for at least 5 min. 4. Fill a large glass tray with transfer buffer and place one of the transfer blotting pads in it. 5. Build a blot sandwich on the sponge: Whatman paper, gel, nitrocellulose, Whatman paper. Carefully layer each piece onto the sandwich. Do not get any bubbles in your sandwich.
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6. Place sandwich into transfer tank. SDS in denaturing gel gives proteins a large net negative charge. Place sandwich such that proteins will move out of the gel and onto the nitrocellulose. Nitrocellulose side of sandwich should face positive pole. 7. Transfer proteins overnight at 0.2 Amp with cooling. 8. Turn off power and remove blot and membrane. 9. After removing the blotted Immobilon-P filter you must first dry it to fix the proteins to the membrane before continuing on to any transillumination or rapid immunodetection procedures to ensure optimum results. There are three drying options. a) Soak the membrane in 100% methanol for 10 sec to drive out the water. Then place the blot on a piece of filter paper. Wait for the methanol to evaporate (~15. Min) b) Place the blot in a vacuum chamber (secure the blot between two sheets of filter paper (~30 min.) c) Incubate the blot at 37oC (~1 hr) d) Place the blot on a lab bench to let it dry at room temperature (~2 hr)
SOLUTIONS:
1. 10x Running buffer Compound Amount/100 ml Amount/1000 ml Tris Base 3.0 g 30 g Glycine 14.4 g 144 g H2O to 1 liter, do not pH 2. Transfer Buffer Final Concentration Stock Solution Amount/100 ml Amount/1000 ml 0.1% SDS 0.1 g 1.0 g 20 % methanol methanol 20 ml 200 ml 1X Running buffer 80 ml 800 ml 3. Methanol
PROTOCOL 1.5: STAINING OF WESTERN BLOTS FOR TOTAL PROTEIN

REFERENCE: D. BICKAR AND P. READ. ANAL. BIOCHEM. 203: 109-115 (1992) INTRODUCTION: Many different procedures have been published for staining total protein on Western blots after transfer. Some employ dyes such as Coomassie Brilliant Blue R250, Amido Black, Ponceau S, Fast green FCF or Eosin Y. Others used colloidal dyes such as India ink or colloidal gold or iron. Finally there are procedures using silver staining or biotinylation. We have found the procedure below to be by far the fastest and easiest. It is also very inexpensive, sensitive, reversible, and applicable to a
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wide variety of proteins. Finally, it works well on both nitrocellulose and PVDF (Immobilon) but not nylon type filters. (Remember that Immobilon filters always need to be prewet in methanol and then water prior to carrying out any procedure using aqueous buffers!). The stain can also be used for staining gels prior to transfer, but in this application is not notably superior to Coomassie Brilliant Blue. Staining 1. Submerge the blot for 30" in the staining solution. (If staining is not obvious, continue up to five minutes) 2. Rinse two or three times with 12 mM HCl to remove unbound dye. Protein will stain a vivid, turquoise-blue with little or no background staining of the filter. On PVDF filters a rinse in 20% ethanol or methanol is necessary to remove all the unbound dye. (Immobilon P filters are more difficult to destain than the original Immobilon filters). 3. Photograph the membrane through an orange filter in white light. Lay a ruler beside the filter so that the location of the molecular weight standards can be readily determined if they fade during the hybridization process. Destaining 1. Soak the blot in 0.5 M NaHCO3 for a few minutes and rinse repeatedly with deionized H2O to remove the bulk of the stain from the protein bands. Not all the dye is removed, but that which remains does not appear to interfere with subsequent antibody detection methods. NOTE: The sensitivity of the stain is about 10 ng of protein.
MATERIALS:
1. 12 mM HCl Compound Amount/100 ml Amount/1000 ml HCl (11.6 M, 36% ) 103 ml 1.03 ml 2. 0.05% CPTS Staining Solution Final Concentration Stock Solution Amount/100 ml Amount/500 ml CPTS (copper 50 mg 250 mg phthalocyanin 3, 4', 4", 4"'tetrasulfonic acid tetrasodium Dissolve salt (Aldrich 24,535-6) 12 mM HCl to volume 12 mM HCL 3. 20% ethanol or methanol (for PVDF filters) Compound Amount/100 ml Amount/1000 ml Ethanol or Methanol 20 ml 200 mg Deionized H2 O to volume 4. 0.5 M NaHCO3 Compound Amount/500 ml Amount/1000 ml NaHCO3 21 g 42 g Deionized H2 O to volume
PROTOCOL 1.6: RAPID IMMUNODETECTION PROTOCOL WITH ENHANCED CHEMILUMINESCENCE DETECTION (ECL)
SOURCE: ECL WESTERN BLOTTING PROTOCOLS, AMERSHAM CORP. 1994 This system is based on the detection of light emission from a secondary antibody linked to horseradish peroxidase which catalyzes the oxidation of luminol. Light emission is detected on x-ray film. Although this system is more expensive and less convenient than the colorimetric alkaline phosphatase detection system, the sensitivity can be incredible. The rapid protocol presented here is essentially that presented in Appendix I of the Amersham technical book on ECL. If time is in short supply the following protocol allows the immunodetection using HRP-labeled antibodies to be completed in just over 2 hours, compared to 4 hours 15 minutes for the standard protocol. If desired, the protocol can be further shortened by also optimizing the primary antibody for a shortened incubation. Immediately following this protocol is a method for optimizing the ECL system before you even run an SDS-PAGE gel. If you are starting from scratch it is highly recommended that you follow that protocol first. PROCEDURE: 1. Electrophoresis and blotting should be done according to your favorite protocol.
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2. Blocking the membrane. Non-specific antibody binding sites on membranes must be blocked before probing to prevent high background and/or spurious binding to specific protein bands. Typical blocking reagents are 5% non-fat dry milk (blotto) or 10% BSA dissolved in phosphate buffered saline (PBS) or Tris buffered saline (TBS) solutions which contain 0.05% to 0.1% Trween 20.. Blocking may be accomplished in as little as 10 minutes or may require overnight at 4oC depending on the antigen/antibody background and the specific membrane being used. Both the blocking times and the optimal tween-20 concentrations have to be determined empirically. 3. During the blocking stage dilute the primary antibody in PBS-T or TBS-T. Note: The optimum dilution of the primary antibody will vary and should be determined for each antibody used. See Protocol 1.8. 4. Rinse the membrane 3 X 15 seconds each in PBS-T or TBS-T. Slightly longer rinses are presumably acceptable. 5. Incubate the membrane in the diluted primary antibody for 1 hour (to overnight) at room temperature with agitation. Note: A further shortening of the immunodetection is possible by increasing the primary antibody concentration. This allows a reduction in the incubation time without compromising sensitivity. However, the optimal increase, which saves time, but does not cause severe background problems, must be optimized for each new antibody. 6. Use PBS-T or TBS-T for the washing buffer. Briefly rinse the membrane using three changes of wash buffer, then wash twice for 10 minutes with fresh changes of washing buffer, at room temperature. As large a volume of wash buffer as possible should be used at each time. As a guide, there should be at least 4 ml per cm2 of membrane present. 7. During the wash steps dilute the HRP-labeled secondary antibody in PBS-T or TBS-T. 8. Incubate the membrane in the diluted secondary antibody for 15 minutes at room temperature with agitation. 9. Wash the membrane as described in step 6. 10. Signal Generation/Detection Read through this whole section before proceeding. It is necessary to work quickly once the blots have been exposed to the detection solutions. All steps can be carried out in a dark room; it is only necessary to switch off the light after step J below. Equipment that is needed includes an X-ray film cassette, a roll of Saran Wrap (other 'cling-films' may not be suitable), a timer and blue-light sensitive autoradiography film, for example Hyperfilm-ECL (RPN 2103). If possible, wear powder-free gloves because powder spots cause blank areas on films. a. Mix an equal volume of detection solution 1 with detection solution 2 to sufficiently cover the blot, in this case 5 ml of solution 1 and 2. b. Place a large piece of Saran Wrap on flat bench. Use clean Kimwipe to smooth out the Saran Wrap so that it is perfectly flat. c. Use forceps to remove the blot from the hybridization tube. Be careful to only clamp the edges of the blot to prevent crushing the lane areas. Crushing sometimes causes high background. d. Remove excess wash buffer by holding the blot vertically and touching one of the lower corners to a small pile of Kimwipes. Wait until all the excess moisture is wicked from the blot. e. Place the blot in the middle of the plastic sheet protector or Saran Wrap with the protein side up. f. Using pipette draw up mixed developing solution and apply solution to the corners and middle of the blot. Add enough so that the entire blot is covered and you can see a positive meniscus. Surface tension will hold the fluid on top. Stop adding the solution if it begins to flow out on the surrounding page protector or Saran Wrap. g. Incubate for 1 minute at room temperature. h. While the sample is incubating put a clean piece of Saran Wrap (big enough to wrap the entire blot in) or page protector on the bench and spread it out with a Kimwipe. i. Using fresh Kimwipes, drain off excess detection buffer as described in Step 3. j. If using Saran Wrap, place the blot protein side down onto the new piece of Saran Wrap. Fold the Saran Wrap over the back of the membrane so the membrane is totally covered, and smooth out air pockets. Ideally the Saran Wrap on the protein side of the blot should remain even and smooth using this procedure. As soon as the membrane is wrapped, flip it over so the protein side faces up, and attach fluorescent markers asymmetrically to the side of the blot so that you can orient the autorad later. Alternatively, you can use the plastic sheet protectors. In this case the membrane can be placed protein side up, then covered, and the fluorescent marker attached to the top of the sheet protectors. These are difficult to clean adequately and should be thrown away after the Western has been developed. k. Place the wrapped blot(s), protein side up, in the film cassette. Work as quickly as possible; minimize the delay between incubating the blots in substrate and exposing them to the film. l. Switch off the lights in the dark room and place a sheet of autoradiography film (for example Hyperfilm-ECL) on top of the blots, close the cassette and expose for 15 seconds to 10 minutes or longer if necessary. m. Interpret results. 11. For a Western blot detected with antibodies of known activity: a. If a previous Western blot resulted in high background, dilute the HRP conjugate at least two-fold (when using biotin-streptavidin system, dilute both biotinylated antibody and streptavidin-HRP by the same amount) for future detections.
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b. If a previous Western blot resulted in non-specific banding, but clear background, dilute the primary antibody two-fold for future detections. Background Correction If a result has background or excess "banding" (cross-reactivity) these methods can be used to produce a result that is acceptable. While these three suggestions can improve signal to noise, the best results are obtained after correct antibody concentrations are determined as described above. 1. Remove the membrane from the plastic wrap and repeat the final washing step. Background and unwanted bands (cross-reaction) are lower affinity reactions and the antibodies will wash off faster than specifically-bound antibodies. Remember to re-incubate the blot in ECL detection reagents before exposing to film. 2. Allow the membrane to stand (not exposing to film) for several minutes; with time the HRP generated signal will decrease. In general, diffuse areas of background will disappear faster than specific signal; resulting in improved signal to noise. 3. Treat membrane with 5-10% H2 O2 in PBS for up to 30 minutes. Hydrogen peroxide treatment can help reduce backgrounds and will not affect the membrane. This treatment may not be suitable for rare antigen detection or weakly binding antibodies. Materials:
1. Primary Antibody 2. Secondary Antibody 3. 1X Phosphate Buffered Saline, pH 7.2 (PBS) Compound Amount/ 1000 ml Deionized H2 O 800 ml NaCl 8 g KCl 0.2 g Na2 HPO4 1.44 g KH2 PO4 0.24 g Adjust pH to 7.2 with HCl. Add deionized H2 O to 1 L. Dispense the solution into aliquots and sterilize them by autoclaving for 20 minutes at 15 lb/sq. in. on liquid cycle. Store at room temperature.
4. PBST (0.1 % SDS) Wash Buffer Compound Amount/1000 ml Amount/4000 ml Polyoxethylene Sorbitan Monolaurate 1 g 4 g (Tween 20) Sigma P-1379 1 X PBS, pH 7.2 to 1000 ml to 4000 ml 5. PBST (0.3 % ) Wash Buffer Compound Amount/1000 ml Amount/4000 ml Polyoxethylene Sorbitan Monolaurate 3 g 12 g (Tween 20) Sigma P-1379 1 x PBS, pH 7.2 to 1000 ml to 4000 ml
6. Tris-Buffered Saline, pH 7.4 (PBS) Compound Amount/ 1000 ml Deionized H2 O 800 ml NaCl 8 g KCl 0.2 g Tris base 3 g Adjust pH to 7.4 with HCl Add deionized H2 O to 1 L Dispense the solution into aliquots and sterilize them by autoclaving for 20 minutes at 15 lb/sq. in. on liquid cycle. Store at room temperature. 7. TBS-T is TBS buffer supplemented with the desired amount of Tween-20 8. 10% non-fat dried milk (Carnation) in PBS
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Compound Amount/100 ml Amount/500 ml Non-fat dried milk 10g 100 g H2 O to 100 ml to 100 ml Stir vigorously on stir plate until dissolved which may take an hour or more. 9. ECL Western Kit with detection reagents 10. Saran Wrap or Page Protectors 11. Immobilon P (Millipore) 12. Plastic Page Protectors 13. 5-10% H2O2 in PBS Compound Amount/100 ml Amount/500 ml H2O2 5 to 10 ml 25 to 50 ml Deionized H2 O to volume Homemade ECL Reagents
Note: Although the majority of workers continue to use the original ECL reagent developed by Amersham, Pierce now sells several alternative formulations which seem to offer the advantages of a longer signal output and lower cost. Pierce also sells an ultrasensitive reagent that is very expensive, but works well. For routine use, you can use "homemade" ECL reagents. Two formulations from the Internet are: 14. Stock Solutions For ECL - Formula 1 250 mM Luminol (Sigma A-8511, 5g) Compound Amount/ 10 ml DMSO 10 ml Luminol 0.44 g\ Store in a dark bottle 90 mM p-Coumaric acid (Sigma C-9008, 10 g - there are smaller sizes) Compound Amount/ 10 ml Amount/ 50 ml DMSO 10 ml 50 ml p-Coumaric acid 0.15 g .75 g 1 M Tris, 8.5 (FW=121.1) Compound Amount/ ml Amount/ ml Deionized H2 O 800 ml Tris base 121.1 g Adjust to pH 8.5 by adding concentrated HCl. Allow the temperature to cool to room temperature before making the final adjustments to the pH. This is because the pH of Tris solutions are temperature-dependent and decrease approximately 0.03 pH for each 1 o C increase in temperature. For example, a 0.05 M solution has pH values of 9.5, 8.9, and 8.5 at 5 o C, 25 o C, and 37 o C respectively. Sterilize by autoclaving.
Solution A Compound Amount/ 100 ml Amount/ 500 ml Luminol Stock 1.0 ml 5.0 ml p-Coumaric acid stock 0.44 ml 2.2 ml 1 M Tris-HCl, pH 8.5 10 ml 50 ml Deionized H2 O to volume. Store at 4 oC under light tight conditions.
Solution B Compound Amount/ 10 ml Amount/ 100 ml 1 M Tris, pH 8.5 1.0 ml 10 ml
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30% H2 O2 6.1 ml 61 ml Deionized H2 O to volume Store at 4 oC, but goes bad quickly so make weekly.
Mix Solutions A and B 1:1 just prior to use. Do not allow any cross-contamination to occur. 15. Stock Solutions for Luminol - Formula 2 Luminol Stock (Sigma A-8511, 5g) Compound Amount/ 10 ml Amount/ 50 ml DMSO 10 ml 50 ml 4-Methylumbellierone 17.7 mg 88.5 mg Store in a dark bottle 100 mM 4-Methylumbelliferone Compound Amount/ 10 ml Amount/ 50 ml DMSO 10 ml 50 ml 4-Methylumbellierone 176 mg 880 mg Combined Solution Compound Amount/ 10 ml Amount/ 50 ml 4-Methylumbelliferone Stock 40 ml 200 ml Luminol Stock 75 ml 375 ml 30% H2 O2 2.8 ml 14.0 ml Deionized H2 O to volume. Make just prior to use. Works reliably and is reproducible, but is slightly less sensitive than the commercial formulations. Alternative Combined Solution Compound Amount/ 10 ml Amount/ 50 ml 4-Methylumbelliferone Stock 40 ml 200 ml Luminol Stock 75 ml 375 ml Sodium perborate 6.15 mg 30.75 mg Deionized H2 O to volume. Make just prior to use. Works reliably and is reproducible, but is slightly less sensitive than the commercial formulations. In our limited experience with the formulation given in 14, we found sensitivity similar to the Amersham ECL reagent. We have not tried formulation 15.
PROTOCOL 1.7: OPTIMIZING ECL WESTERN BLOTTING

SOURCE: AMERSHAM TECHTIP 129 INTRODUCTION: This protocol is designed to provide a guideline for optimizing Western blot detection using the ECL Western blotting detection system (RPN.2106, RPN.2108, RPN.2109). Because ECL is extremely sensitive, determination of optimum antibody concentrations is important to ensure the best results. Generally, less primary and secondary antibody is required for sensitive detection when using ECL compared to colorimetric detection. Reduction in antibody use is one of the many benefits of ECL for Western blot detection. Additional benefits include, (1) greater sensitivity than traditional colorimetric methods; (2) rapid results in seconds to minutes; (3) results on film, hard copy results which do not fade; (4) ability to make multiple copies of results. Determination of Optimum Antibody Concentration The key to sensitive detection is determining the optimal antibody concentrations. While proper blocking and washing are important, they are only temporary measures until the proper antibody concentration is determined. Outlined below are protocols for determining optimal antibody concentrations for the detection of ECL Western blots. 1. For a primary antibody/antiserum with unknown activity, a dot blot is a quick and effective method for determining optimum dilution. Different blocking and washing steps than those offered below may be required for some antibodies. a. Spot approximately 1-5 mg of protein sample on nitrocellulose (e.g., Hybond ECL, RPN.2020D); allow to dry. Prepare one dot blot for each primary antibody dilution to be tested. b. Block for 11/2 hour (e.g. 10% non-fat dried milk in PBS plus 0.1% Tween 20) at room temperature. Wash 3 X 5 min. in 0.1% PBS Tween20. c. Prepare several dilutions of primary antibody: 1/100, 1/500, 1/1000, 1/5000 for instance; incubate 1 blot with each antibody dilution for 1
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hour at room temperature. d. Wash (e.g., PBS plus 0.1% Tween 20) 3x for 5 minutes. e. Dilute secondary antibody; incubate each test blot for 30 min. at room temperature. f. Wash 3x for 5 minutes in 0.3% Tween 20 washes for 5 minutes each. Some weak binding antibodies can be adversely affected by PBS-0.3% Tween 20. In this case 3 X 5 min. washes in PBS--0.1% Tween are recommended. g. Incubate in ECL detection reagents and expose to film as directed in protocol booklet. 2. For a secondary antibody with unknown activity, a dot blot is also effective. a. Spot approximately 1-5 mg of protein sample on nitrocellulose (e.g., Hybond ECL, RPN.2020D); dry at room temperature. Prepare 1 dot blot for each secondary antibody dilution to be tested. b. Block for 1 hour (e.g., 10% non-fat dried milk in PBS plus 0.1% Tween 20) at room temperature. c. Dilute primary antibody; incubate each test blot for 1 hour. d. Wash (e.g., PBS plus 0.1% Tween 20) 3x for 5 minutes. e. Prepare several dilutions of secondary antibody: for example 1/3,000, 1/5,000, 1/7,500, 1/10,000, 1/25,000, 1/50,000; incubate one blot with each dilution for 1 hour at room temperature. f. Wash 3x for 5 minutes each in PBS--0.1% Tween 20 followed by three PBS--0.3% Tween 20 washes for 5 minutes each. Some weak binding antibodies can be adversely affected by PBS--0.3% Tween 20. In this case 6 washes in PBS--0.1% Tween are recommended. g. Do not let the membrane dry out. Keep it moist by capping the tube again until time for signal detection. 3) Perform the detection with ECL reagents as outlined starting on step 3 in Protocol 1.7.
The Materials List Follows Protocol 1.7.
PROTOCOL 1.8: EXPRESSION OF CLONED DNA IN PET PLASMIDS AND DETECTION OF RESULTING PROTEIN
INTRODUCTION: There are a large number of systems available for expressing cloned genes in a variety of hosts. Many of these hosts e.g. yeast or insect cells are capable of producing functionally active mammalian proteins. Bacterial cells are also capable of producing functionally active mammalian proteins, but often bacteriallyexpressed proteins are non-functional either because they lack important post-translational modifications such as phosphorylation or glycosylation and/or because the proteins fold improperly and aggregate. Despite these limitations, bacterial expression systems can still be useful for a variety of studies. For example, they can produce large quantities of protein for antibody production or for use as substrates for post-translational modification enzymes. Expression systems based on the expression of proteins from bacteriophage T7 promoters are especially popular because they can produce extremely high levels of the desired protein amounting to as much as 25% of the total cell protein. The efficiency of these systems derives from the fact that the T7 RNA polymerase is highly selective for transcription from its own promoters. As a result, in the presence of T7 RNA polymerase, nearly all the cells production of proteins comes from the cloned gene. Indeed, the system is so efficient that often the problem is ensuring that there is not premature expression of toxic proteins during the growth of the cells. Because of the problem of premature expression, the developers of the T7 system have developed ways of keeping tight control on the expression of the cloned gene. The first level of control is to control the level of the T7 RNA polymerase itself. This is usually accomplished by placing the polymerase under the control of the highly inducible lacUV5 promoter which can be induced by IPTG. However, even though the basal expression from this promoter is quite low, modest amounts of T7 RNA polymerase can still be expressed in such uninduced cells. To circumvent this low level of expression several procedures have been developed. One uses the presence in the cell of a second plasmid that produces T7 lysozyme-an inhibitor of the polymerase. In another scenario, the cloned gene itself is placed under the control of the lac operator which reduces expression in the absence of IPTG. The Novagen Co. (Madison, WI) has developed a large number of such vectors under the name pET vectors. Many of these vectors have special features that facilitate the purification of the expressed proteins such as affinity tags and protease sensitive sites for removing the affinity tags after purification. In the present protocol, inserts that have been subcloned into the vector pET-28 in the host cell BL21(DE3) are induced with IPTG to provide expression of the polymerase. The gene providing the polymerase is actually contained within a lambda phage DE3 that lysogenizes the host. Proteins from the induced cells are run on SDS/PAGE and the fusion protein identified by its high level of expression and its molecular weight. A more definitive identification of the protein would involve Western blotting to ensure that the proper gene had been cloned and expressed.
PROCEDURE: 1. For screening of protein expression, it is recommended to start with multiple colonies. In some circumstances some colonies may not be expressing as high as the others. Make a 1:100 dilution of an overnight culture of BL21(DE3)pLysS containing your gene cloned into the pET vector into 5 ml of LB with appropriate antibiotics for plasmid selection. 2. Grow at 370 C for 1.5-3 h until the OD600 is about 0.3.
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3. The cultures then need to be split into two halves. Induce one half with IPTG to a final concentration of 0.2 mM. The other half should be left uninduced as a control. 4. Continue the incubation for another 1-4 hours. The time depends on the particular protein. 5. Transfer the cultures into screw-cap or lock-top microfuge tubes and spin them in a microcentrifuge for 1 min. 6. Discard the supernatant and drain the pellets using a Pipetman tip. 7. You can freeze the pellet at -20oC to help lyse cells and to slow degradation. You can leave the pellet frozen indefinitely if your protein of interest is fairly stable. However it is best to immediately proceed to step 8. 8. Immediately after the supernatant has been removed from the cell pellet (or the stored cell pellet has been thawed rapidly), add 50 - 100 ml of sterile water and resuspend cell pellet. You may have to pipette the cells up and down to resuspend the cells. It is important to resuspend the cells before adding the lysis buffer to ensure complete lysis. 9. Immediately add a volume of ice cold 2X sample buffer approximately equal to the estimated volume of the cell pellet plus the water added to resuspend the pellet (~75 to 100 ml). Be sure to keep your final volume between 100-200 ml to keep your protein concentration sufficiently high. 9. Vortex to resuspend the pellet and immediately place the screw cap microfuge or lock top microfuge tube in boiling water bath for 2-5 min to prevent degradation. After boiling, you may again have to pipette up and down to resuspend the denatured proteins. If your prep is very viscous, sonicate the sample with two short 5 sec. bursts at medium power and chill the sample on ice until you are ready to load it on the SDS/PAGE Gel. This treated sample can be stored frozen for future runs. 10. The samples next need to be then analyzed on a SDS/PAGE gel, Protocol 1.3 starting at the section on Sample Loading and Final Assembly of the SE 250.. Usually about 5 ml of sample gives sufficient protein to allow visualization of the bands upon staining. MATERIALS:
1. LB Medium (Luria-Bertani Medium) In a clean dry 2L Erlenmeyer flask add the constituents listed below. If you turn the Bacto-tryptone and Yeast extract bottles on their side and gently tap them to loosen up the contents before you open the bottle, the contents will shift so they can be more easily scooped out of the bottle without generating a large amount of dust. Compound Amount/1000 ml Bacto-tryptone 10 g Yeast extract 5 g NaCl 10 g Add 900 ml H2 O. Stir until the solutes have dissolved. Adjust to pH 7.5 with 5 N NaOH. Adjust the volume of the solution to 1 liter with deionized H2 O. Sterilize by autoclaving for 20 minutes at 15 lb/sq. in. on liquid cycle. 2. Appropriate antibiotics For Vector Being Used Sok o t n tc sl i a uo C net t n o cnr i ao sre ta og Wok g o cnr i rn cnet t n i ao s i et tn n rg p si l md a s A p in m il cl i C reil a n in b cl i C l a p ei l h r hn o om c K nm c aa yi n Sr tm c t po yi e n T t ccnb e ayle r i 5 m /l H 0 0 gm i 2 n 5 m /l H 0 0 gm i 2 n 3 m /l e ao 4 gm i t n l n h 1 m /l H 0 0 gm i 2 n 1 m /l H 0 0 gm i 2 n 5 gm ie ao m /l t n l n h -0 2C 0 -0 2C 0 -0 2C 0 -0 2C 0 -0 2C 0 -0 2C 0 2 m gm 0 /l 2 m gm 0 /l 2 m gm 5 /l 1 m gm 0 /l 1 m gm 0 /l 1 m gm 0 /l rae e xd l p si l md a s 5 m gm 0 /l 5 m gm 0 /l 10 gm 7 m /l 5 t 10 gm 0o 0 m /l 5 m gm 0 /l 5 m gm 0 /l
a Stock solutions of antibiotics dissolved in H2 0 should be sterilized by filtration through a 0.22-micron filter. Antibiotics dissolved in ethanol need not be sterilized. Store solutions in light-tight containers. b Magnesium ions are antagonists of tetracycline. Use media without magnesium salts (e.g., LB medium) for selection of bacteria resistant to tetracycline. 3. 100 mM IPTG (FW = 238.3) Compound Amount/1 ml Amount/10 ml Isopropylthio-b-D-galactosidase 2.38 mg 23.83 mg (IPTG) Deionized H2 O to volume.
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Sterilize by filtration through a 0.22 mm disposable filter. Dispense the solution into 1-ml aliquots and store them at -20 o C. 4. SDS PAGE Gel of appropriate concentration 5. 0.5 M Tris-Cl pH 6.8 Compound Amount/50 ml Amount/100 ml Tris-Cl 3.0 g 6.0 g Deionized H2O 45 ml 95 ml Adjust to pH 6.8 with 1 M HCl. Add deionized H2 O to 50 ml to 100 ml
Allow the temperature to cool to room temperature before making the final adjustments to the pH. This is because the pH of Tris solutions are temperature-dependent and decrease approximately 0.03 pH for each 1 o C increase in temperature. For example, a 0.05 M solution has pH values of 9.5, 8.9, and 8.5 at 5 o C, 25 o C, and 37 o C respectively. Sterilize by autoclaving. 6. 10% Sodium Dodecyl Sulfate Sodium Salt (SDS, FW=288.4, also called Sodium Lauryl Sulfate) Compound Amount/500 ml Amount/1000 ml Deionized H2 O 450 ml 900 ml SDS 50 g 100 g Heat to 68 o C to assist dissolution. Adjust pH to 7.2 by adding a few drops of concentrated HCl. Adjust to volume with deionized H2 O. Dispense in aliquots. There is no need to sterilize 10% SDS. Caution: Wear a mask when weighing SDS and wipe down the weighing area and balance after use because the fine crystals of SDS disperse easily. 7. 2X Sample buffer (Minus Bromophenol Blue) Final Concentration Stock Solution Amount/10 ml Amount/50 ml 0.125 M Tris-Cl pH 6.8 0.5 M Tris-Cl pH 6.8 2.5 ml 12.5 ml 4 % SDS 10% SDS 4.0 ml 20 ml 10% Glycerol 2.0 ml 10 ml 10% 2-Mercaptoethanol 1.0 ml 5 ml H2O 0.5 ml 3.5 ml Divide in aliquots and freeze. 8. Boiling water bath. 9. Sonicator or 1 ml syringe with 19 and 25 gauge hypodermic needles. 10. Appropriate centrifuge tubes and screw cap microfuge tubes. 11. 10 X Bromophenol Blue Compound Amount/1 ml Amount/5 ml Bromophenol Blue 10 mg 50 mg H2 O to 1 ml to 5 ml
PROTOCOL 1.9 RAPID COOMASSIE BLUE STAINING OF PROTEIN GELS

REFERENCE: AUSUBEL ET AL. 10.6.1 INTRODUCTION: There are innumerable variations on the use of Coomassie Brilliant Blue R-250 to stain proteins in gels. Some are faster but less sensitive. In addition there are very sensitive silver staining procedures which are about 100 x as sensitive as the Coomassie procedures. In general, the silver stain procedures take somewhat more hands on time, and require considerable attention to cleanliness. There are also some rapid staining procedures using Cu salts that are intermediate in sensitivity between the Coomassie Blue and silver staining procedures. Ausubel et al. gives additional protocols for doing Coomassie and Silver staining of gels (10.6.1-10.6.2). The following is a rapid Coomassie stain protocol. Because the Coomassie blue concentration is lower than that used in the basic Coomassie staining protocol, the gel background never stains very darkly and the bands can be seen even while the gel remains in the staining solution. Another difference from the basic staining protocol is that isopropanol is substituted for methanol in the fixing and destaining solutions. This protocol is slightly less sensitive than the basic staining protocol. PROCEDURE: 1. Place the polyacrylamide gel in a clean plastic or glass container. Cover the gel with isopropanol fixing solution and shake gently at room temperature for 15 min. (0.7 mm thick gel) to 60 min. (1.5 mm thick gel). 2. Pour out fixing solution. Cover the gel with rapid Coomassie Blue staining solution and shake gently until desired intensity is reached. Bands become visible in 30 minutes and will become more pronounced with longer incubations of 2 h to overnight. A disadvantage of the longer staining times, however, is that the gels require longer to destain.
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3. Pour out staining solution. Cover gel with 10% acetic acid to destain and agitate gently 2 h at room temperature until a clear background is obtained. 4. If a clear background is not obtained, pour out staining solution and repeat step 3. This step is generally unnecessary. Note: You can speed up the destaining somewhat by including a Kimwipe or piece of sponge in the destaining solution to sop up the dye which diffuses from the gel. 5. Store gel in 7% acetic acid or water, or in plastic wrap at 4oC. 6. If desired photograph or dry gel between membranes. MATERIALS:
1. Isopropanol Fixing Solution Final Concentration Stock Solution Amount/1000 ml Amount/2000 ml 25% isopropanol isopropanol 250 ml 500 ml 10% acetic acid acetic acid 100 ml 200 ml Deionized H2 O to volume. 2. Coomassie Brilliant Blue R-250 Stain: (10% acetic acid) Final Concentration Stock Solution Amount/1000 ml Amount/2000 ml 0.012% wt/vol Coomassie 0.12 g 0.24 g Brilliant Blue R-250 (BIO-RAD 161-0406) 10 % Acetic Acid Acetic Acid 100 ml 200 ml Deionized H2 O to volume. 3. Destaining solution Final Concentration Stock Solution Amount/1000 ml Amount/2000 ml 10% acetic acid Acetic acid 100 ml 200 ml Deionized H2 O to volume.
PROTOCOL 1.10 STAINING OF POLYPEPTIDES WITH COOMASSIE BRILLIANT BLUE IN THE PRESENCE OF PICRATE
REFERENCE J.L. STEPHANO ET AL. ANAL. BIOCHEM. 152: 308-313 (1985) INTRODUCTION: Although this method of Coomassie staining is not appreciably more sensitive than standard methods, it is much faster for staining gels lacking detergents e.g. acetic acid urea gels and considerably faster even for gels containing detergents like SDS or Triton. Furthermore one can sometimes stain low molecular weight peptides that are unstained by conventional Coomassie stain., and the stained gels are suitable for densitometry. About the only drawback to this technique is that bottles of picric acid can explode if allowed to completely dry or if certain metal picrate salts are formed. Hence it is prudent to add a little water to the bottles after opening them and wrap them tightly with Parafilm, and to avoid contact with metals. Waste picrate solutions should be washed down the drain with plenty of water. We give here only the procedure of SDS gels which require a washing to remove detergent prior to staining. For other types of gels consult the original reference. Procedure: (for standard Laemmli gels containing SDS) 1. Wash the detergent out of the gels by 3 washes in fixing solution for 20 minutes each. 2. Add the picrate Coomassie stain. The bands should begin to appear in 10 to 15 minutes although very minor bands may take up to several hours to stain. The gels do not overstain even if left in the staining solution overnight. Although the paper reports that destaining is not needed, the staining will be a bit more distinct if following the brief staining period the gels are washed for a half hour or so in water. The gels can then be dried and photographed. The stain can be reused for multiple gels until staining is no longer satisfactory.
MATERIALS:
1. Fixing solution Final Concentration Stock Solution Amount/1000 ml Amount/2000 ml 45% methanol Methanol 450 ml 900 ml 10% acetic acid Acetic Acid 100 ml 200 ml Deionized H2 O to volume 450 ml 900 ml 2. 0.2% Coomassie Brilliant Blue R-250 dissolved in fixing solution
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Compound Amount/250 ml Amount/500 ml Coomassie Brilliant Blue R-250 0.5 g 1.0 g Fixing solution to volume 3. 0.1 M Picric Acid, pH 7.0 (FW=229.1) Compound Amount/ 100 ml Deionized water 75 ml Picric acid 2.291 g Adjust to pH 7.0 with 1M NaOH Add deionized H2 O to 100ml. 4. Picrate Coomassie Staining Solution Compound Amount/120 ml Amount/1250 ml 0.1 M picric acid adjusted to pH 7.0 100ml 1000 ml 0.2% Coomassie Blue R dissolved in fixing solution 25 ml 250 ml
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Unit 1 - Western Blotting

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UNIT 1: WESTERN BLOTTING

UNIT 1: WESTERN BLOTTING

www.med.unc.edu/pmbb/PMBB web site/mbt/WESTERN.htm

UNIT 1: WESTERN BLOTTING

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UNIT 1: WESTERN BLOTTING

UNIT 1: WESTERN BLOTTING

PROTOCOL 1.2: BRADFORD PROTEIN ASSAY (BIO-RAD REAGENTS)

www.med.unc.edu/pmbb/PMBB web site/mbt/WESTERN.htm

UNIT 1: WESTERN BLOTTING

UNIT 1: WESTERN BLOTTING

proe t i r sd eo tnhy rv es neesr u ssh n e e r li t poi iuncs y p e ca s uo e d a.

www.med.unc.edu/pmbb/PMBB web site/mbt/WESTERN.htm

UNIT 1: WESTERN BLOTTING

www.med.unc.edu/pmbb/PMBB web site/mbt/WESTERN.htm

UNIT 1: WESTERN BLOTTING

www.med.unc.edu/pmbb/PMBB web site/mbt/WESTERN.htm

UNIT 1: WESTERN BLOTTING

A. Preparation Of Separating Gel

B. Preparation Of Stacking Gel

PROTOCOL 1.4: PROTEIN TRANSFER FROM ACRYLAMIDE GEL TO MEMBRANE

UNIT 1: WESTERN BLOTTING

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UNIT 1: WESTERN BLOTTING

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UNIT 1: WESTERN BLOTTING

PROTOCOL 1.5: STAINING OF WESTERN BLOTS FOR TOTAL PROTEIN

UNIT 1: WESTERN BLOTTING

www.med.unc.edu/pmbb/PMBB web site/mbt/WESTERN.htm

UNIT 1: WESTERN BLOTTING

UNIT 1: WESTERN BLOTTING

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UNIT 1: WESTERN BLOTTING

Solution B Compound Amount/ 10 ml Amount/ 100 ml 1 M Tris, pH 8.5 1.0 ml 10 ml

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UNIT 1: WESTERN BLOTTING

PROTOCOL 1.7: OPTIMIZING ECL WESTERN BLOTTING

UNIT 1: WESTERN BLOTTING

The Materials List Follows Protocol 1.7.

UNIT 1: WESTERN BLOTTING

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UNIT 1: WESTERN BLOTTING

PROTOCOL 1.9 RAPID COOMASSIE BLUE STAINING OF PROTEIN GELS

UNIT 1: WESTERN BLOTTING

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UNIT 1: WESTERN BLOTTING

www.med.unc.edu/pmbb/PMBB web site/mbt/WESTERN.htm

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