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CL eo|thcore

lure s|mp||c|tg or
togged prote|ns
The journeg rom o torget gene to pur|ned prote|n 3
C$T-togged prote|ns
lntroduct|on to the C$T us|on sgstem 4
pCL vectors 5
^pp||cot|ons 6
Tog c|eovoge enzgmes l3
^pp||cot|ons l4
0etect|on o C$T-togged prote|ns l7
0rder|ng |normot|on l9
|st|d|ne-togged prote|ns
lntroduct|on to h|st|d|ne-togged prote|ns 20
^pp||cot|ons 2l
0rder|ng |normot|on 3l
Strep-tog ll prote|ns
lntroduct|on to Strep-tog ll prote|ns 32
^pp||cot|ons 33
0rder|ng |normot|on 36
l3l-togged prote|ns
lntroduct|on to l3l-togged prote|ns 38
^pp||cot|ons 39
0rder|ng |normot|on 43
0uo|-togged prote|ns
lntroduct|on to duo|-togged prote|ns 44
^pp||cot|ons 45
Contents
The journeg rom o torget
gene to pur|ned prote|n
The use o recomb|nont prote|ns hos |ncreosed greot|g |n recent geors,
os hos the weo|th o techn|ques ond products used or the|r omp||ncot|on
ond pur|ncot|on. The odvontoges o us|ng o recomb|nont togged prote|n
to oc|||tote pur|ncot|on ond detect|on ore now w|de|g recogn|zed. ln
genero|, the nrst step |n o prote|n express|on workfow |nvo|ves c|on|ng o
the torget gene |nto on oppropr|ote express|on vector. Th|s |s o||owed bg
the tronsormot|on o the express|on vector |nto o su|tob|e host sgstem
or subsequent express|on ono|gs|s.
long host sgstems ore ovo||ob|e |nc|ud|ng bocter|o, geost, p|onts,
n|omentous ung|, |nsect or mommo||on ce||s grown |n cu|ture, ond
tronsgen|c on|mo|s or p|onts. Loch host sgstem hos |ts own odvontoges
ond d|sodvontoges, ond |t |s |mportont to cons|der these beore the nno|
se|ect|on o o host. ^ter |n|t|o| screen|ng ond |dent|ncot|on o opt|mum
express|on cond|t|ons or gour port|cu|or prote|n, gou con sco|e-up the
pur|ncot|on process to obto|n |orge omounts o the torget prote|n or
downstreom opp||cot|ons such os unct|ono| ond structuro| stud|es.
The pur|ncot|on o togged prote|ns |s re|ot|ve|g s|mp|e ond soves t|me
due to the h|gh spec|nc|tg between the tog on the expressed prote|n ond
the ||gond on the onn|tg med|um. \ou get h|gh pur|tg |n o s|ng|e step-
onn|tg pur|ncot|on tgp|co||g g|ves up to 95% pur|tg. further pur|ncot|on
steps mog be necessorg | gou requ|re greoter pur|tg.
Cloning Cell culture Screening
Scale-up
purication
Structural
& functional
studies
Lead design
3
4
C$T-togged prote|ns
C|utoth|one $-tronserose |C$Tl Cene fus|on $gstem |s o versot||e
sgstem or the express|on, pur|ncot|on, ond detect|on o C$T-togged
prote|ns produced |n E. coli. Tgp|co| eotures o C$T-togged prote|ns
|nc|ude h|gh b|nd|ng spec|nc|tg to g|utoth|one ||gonds on C|utoth|one
$ephorose chromotogrophg med|o, resu|t|ng |n greoter thon 90%
pur|tg |n one step o the e|uted torget mo|ecu|e. The C$T tog mog
o|so |ncreose the so|ub|||tg ond stob|||tg o the torget prote|n. C$T |s
re|ot|ve|g |orge w|th o re|ot|ve mo|ecu|or moss |l
r
l o 26 000.
^ spec|nc c|eovoge sequence o||ows s|mp|e removo| o the C$T tog
w|th the use o d|erent proteoses oter pur|ncot|on.
lur|ncot|on o C$T-togged prote|ns con be perormed under verg
m||d cond|t|ons, wh|ch preserves the unct|on ond ont|gen|c|tg o the
torget prote|n. C$T togs ore oten used to comp|ement h|st|d|ne togs
or os on o|ternot|ve when h|st|d|ne togs do not g|ve o so|ub|e prote|n
dur|ng express|on.
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pCL vectors produce h|gh
g|e|ds o togged prote|ns
C$T Cene fus|on $gstem |ncorporotes pCL p|osm|ds or
|nduc|b|e, h|gh-|eve| |ntroce||u|or express|on o genes or
gene-rogments os us|ons w|th Schistosoma japonicum
C$T. Lxpress|on |n E. coli g|e|ds togged prote|ns |n the
cgtop|osm w|th the C$T mo|etg ot the om|no term|nus ond
the prote|n o |nterest ot the corboxg| term|nus.
Th|rteen pCL vectors ore ovo||ob|e. N|ne o the vectors
hove on exponded mu|t|p|e c|on|ng s|te |lC$l thot conto|ns
s|x restr|ct|on s|tes. The exponded lC$ oc|||totes the
un|d|rect|ono| c|on|ng o c0N^ |nserts obto|ned rom
||bror|es constructed us|ng mong ovo||ob|e |ombdo vectors.
CL eo|thcore oers pCL vectors w|th encoded recogn|t|on
sequences or s|te-spec|nc c|eovoge bg lre$c|ss|on
lroteose, Thromb|n proteose, ond foctor o proteose |see
pCL vector sequence mop on th|s pogel.
pGEX-1T (27-4805-01)
pGEX-6P-1(27-4597-01)
EcoR I
Sma I
Sal I
Xho I
Not I BamH I
PreScission Protease
Leu Glu Val Leu Phe Gln Gly Pro Leu Gly Ser Pro Glu Phe Pro Gly Arg Leu Glu Arg Pro His
CTG GAA GTT CTG TTC CAG GGG CCC CTG GGA TCC CCG GAA TTC CCG GGT CGA CTC GAG CGG CCG CAT

pGEX-6P-2 (27-4598-01)
BamH I
PreScission Protease
Leu Glu Val Leu Phe Gln Gly Pro Leu Gly Ser Pro Gly Ile Pro Gly Ser Thr Arg Ala Ala Ala Ser
CTG GAA GTT CTG TTC CAG GGG CCC CTG GGA TCC CCAGGA ATT CCC GGG TCG ACT CGA GCG GCC GCA TCG

EcoR I
Sma I
Sal I
Xho I
Not I
pGEX-6P-3 (27-4599-01)
BamH I
PreScission Protease
Leu Glu Val Leu Phe Gln Gly Pro Leu Gly Ser Pro Asn Ser Arg Val Asp Ser Ser Gly Arg
CTG GAA GTT CTG TTC CAG GGG CCC CTG GGA TCC CCG AAT TCC CGG GTC GAC TCG AGC GGC CGC

EcoR I
Sma I
Sal I
Xho I
Not I
BamH I EcoR I
Sma I
Sal I
Xho I
Not I
BamH I EcoR I
Sma I
Sal I
Xho I
Not I
BamH I EcoR I
Sma I
Sal I
Xho I
Not I
BamH I EcoR I
Sma I
Sal I
Xho I
Not I
BamH I EcoR I
Sma I
Sal I
Xho I
Not I
BamH I EcoR I
Sma I
Sal I
Xho I
Not I
EcoR I
CTG GTT CCG CGT GGA TCC CCG GAA TTC ATC GTG ACT GAC TGA CGA
BamH I
Leu Val Pro Arg Gly Ser Pro Glu Phe Ile Val Thr Asp
Thrombin Protease
Stop codons

pGEX
~4900 bp
pBR322
ori
Bal I
BspM I
Ptac
c

a

l
I
q

Nar I
EcoR V
BssH II
BstE II
Mlu I
Apa I
Tth111 I
Aat II
Pst I
p4.5
AlwN I
pSj10 Bam7Stop7
pGEX-4T-2 (27-4581-01)
pGEX-5X-1 (27-4584-01)
pGEX-5X-2 (27-4585-01)
pGEX-5X-3 (27-4586-01)
pGEX-4T-1 (27-4580-01)
pGEX-4T-3 (27-4583-01)
pGEX-3X (27-4803-01)
pGEX-2TK (27-4587-01)
Leu Val Pro Arg Gly Ser Pro Gly Ile Pro Gly Ser Thr Arg Ala Ala Ala Ser
CTG GTT CCG CGT GGA TCC CCA GGA ATT CCC GGG TCG ACT CGA GCG GCC GCA TCG TGA
Stop codon
Ile Glu Gly Arg Gly Ile Pro Glu Phe Pro Gly Arg Leu Glu Arg Pro His Arg Asp
ATC GAA GGT CGT GGG ATC CCC GAA TTC CCG GGT CGA CTC GAG CGG CCG CAT CGT GAC TGA
Stop codons
Ile Glu Gly Arg Gly Ile Pro Gly Ile Pro Gly Ser Thr Arg Ala Ala Ala Ser
ATC GAA GGT CGT GGG ATC CCC GGA ATT CCC GGG TCG ACT CGA GCG GCC GCA TCG TGA
Stop codon
Ile Glu Gly Arg Gly Ile Pro Arg Asn Ser Arg Val Asp Ser Ser Gly Arg Ile Val Thr Asp
ATC GAA GGT CGT GGG ATC CCC AGG AAT TCC CGG GTC GAC TCG AGC GGC CGC ATC GTG ACT GAC TGA
Stop codons
Leu Val Pro Arg Gly Ser Pro Glu Phe Pro Gly Arg Leu Glu Arg Pro His Arg Asp
CTG GTT CCG CGT GGA TCC CCG GAA TTC CCG GGT CGA CTC GAG CGG CCG CAT CGT GAC TGA
Stop codons
Leu Val Pro Arg Gly Ser Pro Asn Ser Arg Val Asp Ser Ser Gly Arg Ile Val Thr Asp
CTG GTT CCG CGT GGA TCC CCG AAT TCC CGG GTC GAC TCG AGC GGC CGC ATC GTG ACT GAC TGA
Stop codons
ATC GAA GGT CGT GGG ATC CCC GGG AAT TCA TCG TGA CTG ACT GAC
Ile Glu Gly Arg Gly Ile Pro Gly Asn Ser Ser
Stop codons
Leu Val Pro Arg Gly Ser Arg Arg Ala Ser Val
Kinase
CTG GTT CCG CGT GGA TCT CGT CGT GCA TCT GTT GGA TCC CCG GGA ATT CAT CGT GAC TGA
Stop codons
Thrombin Protease

Thrombin Protease

Thrombin Protease

Thrombin Protease

Factor Xa Protease

Factor Xa Protease

Factor Xa Protease

Factor Xa Protease

EcoR I BamH I
Sma I
EcoR I BamH I
Sma I
pGEX-2T (27-4801-01)
CTG GTT CCG CGT GGA TCC CCG GGA ATT CAT CGT GAC TGA CTG ACG
Leu Val Pro Arg Gly Ser Pro Gly Ile His Arg Asp
Stop codons
EcoR I
Thrombin Protease

BamH I
Sma I
g
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ta
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e S-transferase
A
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Lven though stop codons |n o|| three romes ore not dep|cted |n
th|s mop, o|| th|rteen vectors hove stop codons |n o|| three romes
downstreom rom the mu|t|p|e c|on|ng s|te.
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lur|ncot|on o
C$T-togged prote|ns
0|erent C|utoth|one $ephorose chromotogrophg med|o ore ovo||ob|e |n servero| ormots. The med|o vorg |n the|r perormonce
porometers, ond the d|erent ormots prov|de opt|ons or sco|e ond conven|ence.
Conto|ns C|utoth|one $ephorose |gh lerormonce
Conto|ns C|utoth|one $ephorose 4 fost f|ow
Conto|ns C|utoth|one $ephorose 43
No
v||| gou use on outomoted pur|ncot|on sgstem such os ^KT^des|gn` \es
C$Tlrep ff l6/l0
Crov|tg-fow co|umns
No
C|utoth|one $ephorose 43
|lrepocked d|sposob|e co|umnl
\es
3otch
No
3u|k C$T lur|ncot|on lodu|e

lrepocked co|umns
C$T $p|nTrop lur|ncot|on lodu|e

C$T lu|t|Trop 43
C|utoth|one $ephorose 43
C$T lu|t|Trop ff
Red|lock C$T lur|ncot|on lodu|e

96-we|| p|ote
$p|n co|umn
C|utoth|one $ephorose 4 fost f|ow
C$Trop ff
$gr|nge
lrepocked co|umns
C$Trop l
C|utoth|one $ephorose |gh lerormonce
\es No
$co|ob|||tg/h|gh fow rotes
lrepocked co|umns
\es
^mount o C$T-togged prote|n
< 50 mg > 50 mg
Glutathione Sepharose
C$Trop 43
|gh reso|ut|on
vhot |s most |mportont to gou`
3uers |nc|uded`
No
\es
* Purication modules also contain buffers
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$creen|ng opt|mo|
cond|t|ons or b|nd|ng
Products featured: GST MultiTrap FF and GST MultiTrap 4B, ExcelGel SDS Gradient 818,
Multiphor II Electrophoresis System, LMW-SDS Marker Kit
lrote|ns ore expressed or unct|ono| ond structuro| stud|es ond one port o the eor|g screen|ng phose |s to nnd out opt|mo| cond|t|ons
or b|nd|ng ond e|ut|on o the torget prote|n.
The eect o d|erent |ncubot|on t|mes or the b|nd|ng o o C$T-togged prote|n somp|e wos |nvest|goted to och|eve the best g|e|d on
C$T lu|t|Trop ff ond C$T lu|t|Trop 43 96-we|| n|ter p|otes. E. coli conto|n|ng C$T-h|ppoco|c|n wos chem|co||g |gsed ond son|coted
pr|or to opp||cot|on o the unc|or|ned somp|e d|rect|g to the we||s.
96-well lter plates: C$T lu|t|Trop ff ond C$T lu|t|Trop 43
Sample: 300 | o E. coli 3L2l |gsote conto|n|ng C$T-togged h|ppoco|c|n,
l
r
45 000
Sample preparation: Chem|co| |gs|s ond son|cot|on
Binding buffer: l0 ml sod|um phosphote, l40 ml NoC|, p 8.0
Elution buffer: 50 ml Tr|s-C|, l0 ml reduced g|utoth|one, p 8.0
Elution method: Centr|ugot|on
SDS-PAGE
500
450
400
350
300
250
200
0 20 40 60 80 100 120 140
Yield GST-hippocalcin, GST MultiTrap FF
Yield GST-hippocalcin, GST MultiTrap 4B
Incubation time (min)
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GST-hippocalcin
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120 min 60 min 30 min 10 min 3 min 0 min
Summary
The opt|mum t|me determ|ned or the b|nd|ng o C$T-h|ppoco|c|n wos 3 to l5 m|n w|th both C$T lu|t|Trop ff ond
C$T lu|t|Trop 43. The g|e|d wos ~350 g us|ng both 96-we|| p|otes.
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$o|ub|||zot|on screen|ng
stroteg|es or C$T-togged
membrone prote|ns
Products featured: GST SpinTrap Purication Module, GST Detection Module
$o|ub|||zot|on |s one o the most cr|t|co| stoges o the extroct|on o membrone prote|ns. Lect|ve so|ub|||zot|on ensures h|gh g|e|ds o
b|o|og|co||g oct|ve membrone prote|n ond poves the wog or successu| pur|ncot|on. for eect|ve so|ub|||zot|on o o torget membrone
prote|n, |t |s oten necessorg to screen severo| detergents beore se|ect|ng the best detergent or o port|cu|or torget prote|n ond |ts
pur|ncot|on strotegg.
C$T $p|nTrop co|umns were used or rop|d, s|mu|toneous screen|ng o s|x d|erent detergents or o C$T-membrone prote|n |C$T-ecoKchl.
96-well plate
l. The eect o d|erent detergents on the enzgmot|c
oct|v|tg o o pur|ned C$T-prote|n wos meosured |n o
l-ch|oro-2,4-d|n|trobenzene |C0N3l ossog |n o 96-we||
p|ote ormot us|ng C$T 0etect|on lodu|e.
2. The membrone prote|ns were so|ub|||zed |n d|erent
detergents ond concentrot|ons thot do not oect the
oct|v|tg o the C$T tog.
3. The so|ub|||zed C$T-togged membrone prote|ns were
pur|ned us|ng C$T $p|nTrop lur|ncot|on lodu|e.

^no|gze g|e|d bg $0$-l^CL ond
oct|v|tg o membrone prote|n.
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Column: C$T $p|nTrop
Sample: 500 | o C$T-togged ecoKch so|ub|||zed l.l0 |w/vl |n
5% 00l, 0C, C^l$, Tween 20, Tr|ton -l00, or $orcosg|
Binding buffer: l3$, p 7.5
Elution buffer: l3$, 0.2% detergent, l0 ml reduced g|utoth|one, p 8.0
SDS-PAGE
DDM
OG
CHAPS Tween 20
Triton
X-100 Sarcosyl
GST-ecoKch
94
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43
(M
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3
)
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F
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f
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G
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S
p
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T
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a
p
0.8
0.6
0.4
0.2
0
Solubilized material
Flowthrough
Eluate
A
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y

(
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4
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*
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D
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O
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A
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2
0
T
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X
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0
0
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Summary
The g|e|d o C$T-ecoKch |n the so|ub|||zot|on |doto not shownl ond pur||cot|on steps wos h|ghest w|th 00l ond sorcosg|. Note
thot sorcosg| greot|g decreoses C$T oct|v|tg |n C0N3 ossogs thereore the resu|ts do not re|ect the true omounts o C$T-togged
prote|n present. Lnzgme oct|v|tg oter pur||cot|on wos h|ghest w|th 00l. Th|s shows o s|mp|e ond rop|d method or se|ect|ng
opt|mum cond|t|ons or membrone prote|n so|ub|||zot|on.
Acknowledgements: 0. 3|rse, 0eportment o 3|ochem|strg ond 3|ophgs|cs, $tockho|m Un|vers|tg, $weden
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Lnc|ent one-step pur|ncot|on
Products featured: GSTrap FF, KTAexplorer, ExcelGel SDS Gradient 818, Multiphor II Electrophoresis System,
LMW-SDS Marker Kit
lur|ncot|on o C$T-togged prote|ns con oten be och|eved |n o s|ng|e step us|ng conven|ent prepocked co|umns comb|ned w|th
o sgr|nge, pump, or chromotogrophg sgstem. ln th|s exomp|e, o C$Trop ff l m| co|umn wos used to pur|g o C$T-togged
so|ub|e receptor subun|t to o h|gh degree o pur|tg |n o s|ng|e step. ^ preprogrommed UNlC0RN method temp|ote wos used
|n ^KT^exp|orer to prov|de o stondord pur|ncot|on protoco| thot con e|ther be o||owed exoct|g or mod|ned to su|t gour un|que
requ|rements. The e|uted roct|on wos ono|gzed bg ge| e|ectrophores|s o||owed bg s||ver sto|n|ng.
Column: C$Trop ff l m|
Sample: 8 m| o cgtop|osm|c extroct rom E. coli express|ng o C$T-togged prote|n
Binding buffer: l3$, p 7.3
Elution buffer: 50 ml Tr|s-C|, l0 ml reduced g|utoth|one, p 8.0
Flow: l m|/m|n
System: ^KT^exp|orer
SDS-PAGE
0
20
40
60
80
100
%
Elution
buffer
0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
ml
Wash
Elution
buffer
2.7 mg
pure
GST-
tagged
protein
5.0 10.0 15.0 20.0
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f
r
a
c
t
i
o
n
Summary
C$Trop ff ond o preprogrommed method temp|ote |n ^KT^exp|orer were used to produce h|gh|g pure prote|n |n o s|ng|e
pur||cot|on step.
G
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p
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s
ll
Summary
The odd|t|on o o second pur||cot|on step eect|ve|g removed oggregotes rom the C$T-h|ppoco|c|n somp|e resu|t|ng |n the
recoverg o h|gh|g pure torget prote|n.
Unottended two-step
outomoted pur|ncot|on
Products featured: GSTrap 4B, HiLoad 16/60 Superdex 200 pg, KTAxpress, ExcelGel SDS Gradient 818,
Multiphor II Electrophoresis System, LMW-SDS Marker Kit
^ter the |n|t|o| pur|ncot|on o o C$T-togged prote|n us|ng C|utoth|one $ephorose products, ree cgste|ne groups on the C$T tog con
reoct w|th eoch other to couse oggregotes desp|te the presence o o reduc|ng ogent such os d|th|othre|to| |00Tl.
Runn|ng o second pur|ncot|on step us|ng ge| n|trot|on chromotogrophg prov|des on enc|ent method or seporot|ng prote|n oggregotes
rom the oct|ve torget prote|n. ^dd|t|ono| benents o such o po||sh|ng step |nc|ude the removo| o other contom|nonts ond buer exchonge.
^KT^xpress prov|des on outomoted so|ut|on or unottended, mu|t|step pur|ncot|on o onn|tg-togged prote|ns. ^ggregotes o C$T-
h|ppoco|c|n were successu||g removed us|ng on outomoted two-step pur|ncot|on method temp|ote |n ^KT^xpress. The two-step method
compr|sed onn|tg chromotogrophg |^Cl ond ge| n|trot|on |Cfl pur|ncot|on. The g|e|d o e|uted C$T-h|ppoco|c|n wos meosured bg
obsorbonce ot 280 nm ond the pur|tg wos ono|gzed bg ge| e|ectrophores|s.
Columns: C$Trop 43 l m| |^Cl ond |Lood l6/60 $uperdex 200 pg, l20 m| |Cfl
Sample: C|or|ned E. coli |gsote conto|n|ng expressed C$T-h|ppoco|c|n, l
r
43 000
Sample volume: 5 m|
Binding buffer (AC): l0 ml sod|um phosphote, l40 ml NoC|, 20 ml 0TT, p 7.4
Elution buffer (AC): 50 ml Tr|s-C|, 20 ml g|utoth|one, 20 ml 0TT, p 8.0
Buffer (GF): l0 ml sod|um phosphote, l40 ml NoC|, 20 ml 0TT, p 7.4
Flow rates: $omp|e |ood|ng, 0.3 m|/m|n wosh ond e|ut|on, l m|/m|n |^Cl l.5 m|/m|n |Cfl
System: ^KT^xpress
SDS-PAGE
500
0
1000
1500
mAU
0 0 2 0 5 150 100
AC GF
ml
A
280
Large aggregates of
GST-hippocalcin
Dimers of GST-hippocalcin
150 140 130 120 110 ml
50
0
100
150
200
250
mAU
A
280
97
66
45
30
20
14
(M
r
10
3
)
GST-hippocalcin
L
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W

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m
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i
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F
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o
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o
u
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h

(
A
C
)
E
l
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d

f
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a
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n
s

o
f

d
i
m
e
r

(
G
F
)
G
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a
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e
d

p
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o
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e
i
n
s
l2
lncreos|ng the pur|ncot|on sco|e
Products featured: pGEX-2T, GSTrap FF, GSTPrep FF 16/10, KTAexplorer, ExcelGel SDS Gradient 818,
Multiphor II Electrophoresis System, LMW-SDS Marker Kit
0nce o method or the pur|ncot|on o o torget prote|n hos been estob||shed, |t con be sco|ed up to produce |orger quont|t|es o
torget prote|n or unct|ono| ond structuro| stud|es. The one-step pur|ncot|on method be|ow |||ustrotes o 26-o|d sco|e-up. The mo|n
porometer |n th|s sco|e-up studg wos res|dence t|me |the per|od o t|me the somp|e |s |n contoct w|th the chromotogrophg med|uml.
Res|dence t|me wos the some or the C$Trop ff l m| ond 5 m| co|umns, but tw|ce os |ong or the C$Tlrep ff l6/l0 |20 m| co|umnl
due to the d|erence |n co|umn |ength ond d|ometer. The g|e|d wos determ|ned bg meosur|ng the obsorbonce ot 280 nm ond pur|tg
wos ossessed bg $0$-l^CL.
Columns: C$Trop ff l m|, C$Trop ff 5 m|, ond
C$Tlrep ff l6/l0 |20 m|l
Sample: C$T-0em^ |n E. coli extroct
Sample volumes: l0 m| |l m| co|umnl, 50 m|
|5 m| co|umnl, 200 m| |20 m| co|umnl
Binding buffer: l3$, p 7.4
Elution buffer: 50 ml Tr|s-C|, l0 ml reduced
g|utoth|one p 8.0
Flow rates: 0.5 m|/m|n ot somp|e |ood|ng, l m|/m|n
ot wosh|ng ond e|ut|on |C$Trop ff l m|l
2.5 m|/m|n ot somp|e |ood|ng, 5 m|/m|n ot wosh|ng
ond e|ut|on |C$Trop ff 5 m|l
5 m|/m|n ot somp|e |ood|ng, l0 m|/m|n ot wosh|ng
ond e|ut|on |C$Tlrep ff l6/l0l
System: ^KT^exp|orer l00
SDS-PAGE
Summary
^n |ncreosed co|umn vo|ume ond somp|e |ood |ed to o proport|ono||g |ncreosed g|e|d o the e|uted C$T-togged prote|n |n the
sco|e-up protoco|s.
mAU
1200
1000
800
600
400
200
0
A
280
Elution buffer
100
80
60
40
20
0
%B
0
Wash
Elution
buffer
5.5 mg
10 20 30 ml
1400
1200
1000
800
600
400
200
0
A
280
100
80
60
40
20
0
%B
Wash
Elution
buffer
20.9 mg mAU
0 50 100 150 ml
Elution buffer
Elution
buffer
A
280
mAU
1400
1200
1000
800
600
400
200
0
Elution buffer
100
80
60
40
20
0
%B
Wash
111.0 mg
0 100 200 300 400 500 600 ml
GST-DemA
97
66
45
30
20
14
(M
r
10
3
)
L
M
W

m
a
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k
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s
S
t
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t

m
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t

m
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F
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F
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E
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f
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t

m
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F
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o
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E
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d

f
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t
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n
s
GSTrap FF 1 ml GSTrap FF 5 ml GSTPrep FF 16/10
GSTrap FF 1 ml GSTrap FF 5 ml GSTPrep FF 16/10
Removo| o |orge prote|n togs such os the C$T tog |s oten necessorg or torget prote|n chorocter|zot|on. The omount o
proteose, temperoture, ond |ength o |ncubot|on requ|red or comp|ete d|gest|on vor|es occord|ng to the noture o the
port|cu|or torget prote|n.
lre$c|ss|on lroteose |s on enc|ent enzgme or the spec|nc c|eovoge ond removo| o C$T togs. lre$c|ss|on lroteose |s
bg |tse| o togged prote|n o C$T ond humon rh|nov|rus 3C proteose, hence |t |s eos||g removed rom the c|eoved torget
prote|n |o|ong w|th C$Tl us|ng C|utoth|one $ephorose onn|tg med|um. $|nce lre$c|ss|on lroteose |s opt|mo||g oct|ve ot
4C, c|eovoge con be perormed ot |ow temperotures to enhonce the stob|||tg o the torget prote|n.
Un||ke other proteoses, the recogn|t|on s|te o lre$c|ss|on lroteose |s not o noturo||g occurr|ng sequence ond th|s coners
o h|gh degree o spec|nc|tg on the enzgme. C|eovoge o C$T-togged prote|ns con o|so be perormed w|th other proteoses
thot recogn|ze d|erent c|eovoge s|tes. foctor o proteose ond Thromb|n proteose ore ser|ne proteoses w|th opt|mo|
c|eovoge perormonce ot room temperoture.
Tog c|eovoge enzgmes
Cleavage site
Glutathione
GST
Recombinant
protein
Sepharose
Cleavage enzymes
Factor Xa protease: l
r
28 000 - 30 000
Thrombin protease: l
r
37 000
PreScission Protease: l
r
46 000
G
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t
a
g
g
e
d

p
r
o
t
e
i
n
s
l3
G
S
T
-
t
a
g
g
e
d

p
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o
t
e
i
n
s
l4
0pt|m|zot|on o tog c|eovoge
Products featured: GSTrap FF, PreScission Protease, KTAxpress
^n opt|mo| cond|t|on or c|eovoge wos enc|ent|g |dent|ned bg screen|ng exper|ments w|th on-co|umn c|eovoge o o C$T-togged
prote|n us|ng C$Trop ff ond lre$c|ss|on lroteose on on ^KT^xpress sgstem. lncubot|on t|me ond the rot|os o proteose ond
togged prote|n were vor|ed. The percentoge o c|eoved prote|n wos determ|ned bg |ntegrot|ng the oreo o the the e|uted, c|eoved
prote|n ond d|v|d|ng th|s w|th the |ntegroted toto| peok oreo |e|uted c|eoved prote|n + unc|eoved prote|nl. The c|eovoge reoct|on
wos perormed ot 4C.
Summary
The opt|m|zed cond|t|ons |dent||ed were 20 U o proteose/mg o torget prote|n or o toto| |ncubot|on t|me o 8 h. Th|s opt|mo|
cond|t|on wos used or urther stud|es.
0
10
20
30
40
50
60
70
80
90
100
10 units PreScission/mg protein
20 units PreScission/mg protein
40 units PreScission/mg protein
2 4 6 8 10 12 14
Incubation time (h)
C
l
e
a
v
e
d

p
r
o
t
e
i
n


(
%
)
0
G
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t
a
g
g
e
d

p
r
o
t
e
i
n
s
l5
lur|ncot|on ond
on-co|umn c|eovoge
Products featured: GSTrap FF, Thrombin protease, KTAexplorer, ExcelGel SDS Gradient 818, Multiphor II
Electrophoresis System, LMW-SDS Marker Kit
C|eovoge o the C$T onn|tg tog con e|ther be perormed on-co|umn beore e|ut|on or |n so|ut|on oter e|ut|on o the torget mo|ecu|e.
To demonstrote the enc|encg o on-co|umn c|eovoge |n conjunct|on w|th pur|ncot|on, o C$T-togged prote|n conto|n|ng the
recogn|t|on sequence or Thromb|n proteose wos opp||ed to o C$Trop ff l m| co|umn. ^ter |ncubot|on w|th the proteose or l6 h ot
room temperoture, the C$T-ree torget prote|n wos e|uted.
SDS-PAGE
Summary
Lect|ve on-co|umn c|eovoge o the C$T mo|etg w|th Thromb|n proteose con be |ntegroted w|th the C$T pur||cot|on process.
Column: C$Trop ff l m|
Sample: l0 m| o c|or|ned cgtop|osm|c extroct rom E. coli express|ng o C$T-togged prote|n
Binding buffer: l3$, p 7.3
Elution buffer: 50 ml Tr|s-C|, l0 ml reduced g|utoth|one, p 8.0
Flow rate: l m|/m|n
System: ^KT^exp|orer l0

0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
5.0 10.0 15.0 min
Wash
Incubation
16 h
room temp.
Wash
A280
Target
protein
Free
GST
2.0 4.0 6.0 8.0 10.0 12.0 min
0
20
40
60
80
100
%
Elution
buffer
97
66
45
30
20
14
(M
r
10
3
)
L
M
W

m
a
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k
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t

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d

p
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,

1

m
l

c
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p
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,

5

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1
6

h

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C
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t
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T
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i
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p
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(
2
0

U
/
m
l
)
L
M
W

m
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s
Thrombin
protease
cleavage,
GSTrap FF 1 ml
Target
protein
G
S
T
-
t
a
g
g
e
d

p
r
o
t
e
i
n
s
l6
^utomoted mu|t|step
pur|ncot|on ond tog removo|
Products featured: pGEX-6P, GSTrap HP, PreScission Protease, HiLoad 16/60 Superdex 75 pg, KTAxpress,
ExcelGel SDS gradient 818, Multiphor II Electrophoresis System, LMW-SDS Marker Kit
C$T-togged prote|ns produced w|th o lre$c|ss|on lroteose c|eovoge s|te enob|e two-step pur|ncot|on w|th on-co|umn tog c|eovoge
|n the nrst onn|tg step. ^ C$T-togged mode| prote|n wos pur|ned w|th ^KT^xpress us|ng on outomoted two-step pur|ncot|on method.
C|eovoge ond removo| o the C$T tog wos |mp|emented |n the protoco|.
Summary
Tog c|eovoge ond removo| were |ncorporoted |nto the outomoted two-step pur||cot|on protoco| ond th|s produced o h|gh|g
pure ond c|eoved torget prote|n.
Columns: ^nn|tg chromotogrophg |^Cl, C$Trop l 5 m|,
Ce| n|trot|on |Cfl, |Lood l6/60 $uperdex 75 pg
Sample: C$T-pur |l
r
6l 600l
Binding/cleavage buffer (AC): 50 ml Tr|s-C|, l50 ml NoC|, l ml L0T^,
l ml 0TT, p 7.5
Elution buffer (AC): 50 ml Tr|s-C|, l0 ml reduced g|utoth|one, p 8.0
Buffer (GF): 50 ml Tr|s-C|, l50 ml NoC|, p 7.5
System: ^KT^xpress
SDS-PAGE
AC GF
0
500
1000
1500
2000
mAU
200 250 300
46 mg
Cleaved protein
Regeneration
A
280
ml
97
66
45
30
20
14
(M
r
10
3
)
L
M
W

m
a
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k
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S
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t

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F
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h
P
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d
,

c
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a
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d

G
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T
-
p
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U
n
c
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a
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d

G
S
T
-
p
u
r

pur
G
S
T
-
t
a
g
g
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d

p
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o
t
e
i
n
s
l7
0etect|on o
C$T-togged prote|ns
lrote|n |mmunodetect|on |s o detect|on method thot meosures o spec|nc ont|gen/ont|bodg reoct|on. C$T 96-we|| 0etect|on lodu|e
mokes |t poss|b|e to v|suo||ze ond quont|tote C$T-togged prote|ns rom o comp|ex prote|n somp|e. The wog o recomb|nont prote|n
o|ds con somet|mes mosk some o |ts b|nd|ng s|tes dur|ng ont|bodg detect|on o C$T-togged prote|ns. The use o on ont|-C$T po|gc|ono|
ont|bodg copob|e o recogn|z|ng more thon one ep|tope on C$T-togged prote|ns greot|g enhonces the chonces o detect|on.
4 5 6 7 8 9 10 11 12 1 2 3
Control Anti-luciferase detection
Columns
l-3 $er|o| d||ut|ons o contro| C$T-|uc|erose
4-l2 50 | o c|or|ned |gsotes rom se|ected co|on|es
ldent|ncot|on o c|ones
express|ng C$T-togged prote|n
Products featured: pGEX-6P-1, GST 96-well Detection Module
0nce the gene o |nterest hos been c|oned |nto the pCL express|on vector ond the host ce||s used or the c|on|ng step hove been
tronsormed, chem|co||g |nduc|b|e h|gh-|eve| express|on o the torget prote|n |s poss|b|e. The next step |s to opt|m|ze C$T-togged
prote|n express|on ond o keg step |s the copob|||tg to screen |gsotes rom mong c|ones.
Cu|tures o rondom|g se|ected E. coli co|on|es resu|t|ng rom o pCL-6l-l/|uc|erose gene c|on|ng exper|ment were grown, |nduced, ond
|gsed |n o 96-we|| p|ote. Coptured C$T-togged |uc|erose detected w|th robb|t ont|-|uc|erose, ont|-robb|t lgC/perox|dose conjugote
us|ng C$T 96-we|| 0etect|on lodu|e. Tl3 |3,3,5,5-tetromethg| benz|d|nel wos used os substrote ond the obsorbonce wos reod ot 450 nm.
Summary
f|tg-three percent o the c|ones tested expressed C$T-togged |uc|erose.
l8
G
S
T
-
t
a
g
g
e
d

p
r
o
t
e
i
n
s
SDS-PAGE
97
66
45
30
20
14
(M
r
10
3
)
S
o
n
i
c
a
t
e
,

E
.

c
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1
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4
5

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,

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,

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c
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+

p
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-
5
X
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g

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-
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,

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+

p
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-
4
T
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7

e
x
p
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s
i
n
g

G
S
T
-
E
7
P
u
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i
f
i
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d

G
S
T
P
r
e
s
t
a
i
n
e
d

M
W

m
a
r
k
e
r
vestern b|ot detect|on
Products featured: Anti-GST Antibody, Amersham Hybond ECL, Amersham Hyperlm ECL, Amersham GST
Western Blotting Detection Kit
$omet|mes, E. coli ce||s mog hove d|ncu|tg overexpress|ng o port|cu|or torget prote|n. To test the encocg o the express|on sgstem,
E. coli ce||s conto|n|ng o pCL-5-l vector w|thout on |nsert, were |nduced to overexpress the C$T mo|etg on|g. Two d|erent C$T-togged
prote|ns were then overexpressed |n E. coli ond detected bg the use o ^nt|-C$T ^nt|bodg ond vestern b|ot us|ng LCL detect|on.
HRP
Summary
^nt|-C$T ^nt|bodg prov|des o qu|ck ond conven|ent method or detect|ng C$T-togged prote|ns.
L|ght
$econdorg ont|bodg
Rl conjugote
lr|morg ont|bodg
^nt|gen on membrone
LCL detect|on reogent
l9
0rder|ng |normot|on
Product Quantity Code No.
Protein expression
pCL-2T 25 g 27-480l-0l
pCL-2TK 25 g 27-4587-0l
pCL- 4T-l 25 g 27-4580-0l
pCL- 4T-2 25 g 27-458l-0l
pCL- 4T-3 25 g 27-4583-0l
pCL-3 25 g 27-4803-0l
pCLlT 5 g 27-4805-0l
pCL- 5-l 25 g 27-4584-0l
pCL- 5-2 25 g 27-4585-0l
pCL- 5-3 25 g 27-4586-0l
pCL- 6l-l 25 g 27-4597-0l
pCL- 6l-2 25 g 27-4598-0l
pCL- 6l-3 25 g 27-4599-0l
All vectors include L. co|| BL21
Purication
C$Trop l 5 l m| l7-528l-0l
l00 l m|

l7-528l-05
l 5 m| l7-5282-0l
5 5 m| l7-5282-02
l00 5 m|

l7-5282-05
C$Trop ff 2 l m| l7-5l30-02
5 l m| l7-5l30-0l
l00 l m|

l7-5l30-05
l 5 m| l7-5l3l-0l
5 5 m| l7-5l3l-02
l00 5 m|

l7-5l3l-05
C$Tlrep ff l6/l0 l 20 m| l7-5234-0l
C$T lu|t|Trop ff 4 96-we|| n|ter
p|otes
28-4055-0l
C$Trop 43 5 l m| 28-40l7-45
l00 l m|

28-40l7-46
l 5 m| 28-40l7-47
5 5 m| 28-40l7-48
l00 5 m|

28-40l7-49
C$T $p|nTrop
lur|ncot|on lodu|e
50 50 | 27-4570-03
C$T lu|t|Trop 43 4 96-we|| n|ter
p|otes
28-4055-00
C$T 0etect|on lodu|e 50 detect|ons 27-4590-0l
C$T 96-ve|| 0etect|on
lodu|e
5 p|otes 27-4592-0l
Detection
^nt|-C$T ^nt|bodg 0.5 m|,
50 detect|ons
27-4577-0l
^mershom LCL C$T vestern
3|ott|ng 0etect|on K|t
l k|t RlNl237
^mershom gpern|m LCL
|l8 x 24 cml
50 sheets 28-9068-36
^mershom gbond LCL
|20 20 cml
l0 sheets RlN20200
Tag cleavage
Thromb|n proteose 500 un|ts 27-0846-0l
foctor o proteose 400 un|ts 27-0849-0l
lre$c|ss|on lroteose 500 un|ts 27-0843-0l
Related products
C|utoth|one $ephorose |gh
lerormonce
25 m| l7-5279-0l
l00 m| l7-5279-02
C|utoth|one $ephorose
4 fost f|ow
25 m| l7-5l32-0l
l00 m| l7-5l32-02
500 m| l7-5l32-03
C|utoth|one $ephorose 43 l0 m| l7-0756-0l
l00 m|
|unct|on testedl
27-4574-0l
300 m| l7-0756-04
3u|k C$T lur|ncot|on lodu|e l k|t 27-4570-0l
Red|lock C$T lur|ncot|on
lodu|e
l k|t 27-4570-02
C|utoth|one $ephorose 43
|prepocked d|sposob|e
co|umnsl
2 x 2 m| l7-0757-0l
|Lood l6/60 $uperdex 30 pg l l20 m| l7-ll39-0l
|Lood 26/60 $uperdex 30 pg l 320 m| l7-ll40-0l
|Lood l6/60 $uperdex 75 pg l l20 m| l7-l068-0l
|Lood 26/60 $uperdex 75 pg l 320 m| l7-l070-0l
|Lood l6/60 $uperdex 200 pg l l20 m| l7-l069-0l
|Lood 26/60 $uperdex 200 pg l 320 m| l7-l07l-0l
Lxce|Ce| $0$
Crod|ent 8-l8
6 80-l255-53
Llv-$0$ lorker K|t l0 v|o|s l7-0446-0l
Product Quantity Code No.

available by specic customer order


For more information on equipment for chromatography and/or electrophoresis, please visit www.gelifesciences.com
G
S
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|st|d|ne-togged prote|ns
|st|d|ne togs ore w|de|g used becouse theg ore smo|| ond rore|g |nterere
w|th the unct|on, oct|v|tg, or structure o torget prote|ns. lmmob|||zed meto|
|on onn|tg chromotogrophg |ll^Cl |s the most common method or
pur|g|ng h|st|d|ne-togged prote|ns. ll^C chromotogrophg med|o chorged
w|th d|vo|ent meto| |ons such os n|cke| se|ect|ve|g reto|n h|st|d|ne-togged
prote|ns ond o||ow or the pur|ncot|on o |nso|ub|e h|st|d|ne-togged prote|ns
rom |nc|us|on bod|es when denotur|ng cond|t|ons ore used. $uccessu|
ll^C pur|ncot|on g|ves o h|gh g|e|d o pure ond oct|ve torget prote|n.
owever, s|nce mong prote|ns hove |ntr|ns|c h|st|d|ne ond/or cgste|ne
om|no oc|d res|dues, other nonspec|nc prote|ns b|nd to the ll^C med|o
together w|th the torget prote|n. ln such coses, |t |s oten necessorg to
opt|m|ze b|nd|ng, wosh, ond e|ut|on cond|t|ons bg vorg|ng the concentrot|on
o |m|dozo|e |n these so|ut|ons. lncreos|ng the concentrot|on o |m|dozo|e
|n the b|nd|ng ond wosh buers genero||g decreoses nonspec|nc b|nd|ng,
whereos |ower concentrot|ons g|ve stronger onn|tg |nteroct|on. The keg |s
nnd|ng the r|ght bo|once.
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lur|ncot|on o h|st|d|ne-
togged prote|ns
0|erent N| $ephorose chromotogrophg med|o ore ovo||ob|e |n servero| ormots. The med|o vorg |n the|r perormonce porometers,
ond the d|erent ormots prov|de opt|ons or sco|e ond conven|ence.
Conto|ns N| $ephorose |gh lerormonce
Conto|ns N| $ephorose 6 fost f|ow
No
v||| gou use on outomoted pur|ncot|on sgstem such os ^KT^des|gn` \es
|slrep ff l6/l0
Crov|tg-fow co|umns
No
|s Crov|Trop
No
3otch
\es
|s Crov|Trop K|t
vhot ore gour requ|rements`
3uers |nc|uded`
|s $p|nTrop
|s lu|t|Trop ff
N| $ephorose 6 fost f|ow
|s lu|t|Trop l
96-we|| p|ote
$p|n co|umn
lrepocked co|umns
|sTrop l
N| $ephorose |gh lerormonce
\es No
$co|ob|||tg/h|gh fow rotes
lrepocked co|umns
\es
^mount o h|st|d|ne-togged prote|n
> 200 mg < 200 mg
Ni Sepharose
|gh reso|ut|on
$gr|nge
3uers |nc|uded`
|sTrop ff crude
No \es
|sTrop ff crude K|t
|sTrop ff
C|or|ned or unc|or|ned somp|e
Unc|or|ned C|or|ned
|s $p|nTrop K|t
3uers |nc|uded`
\es No
2l
22
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^utomot|on o h|gh-throughput
express|on screen|ng
Products featured: His MultiTrap HP
^utomoted ond reproduc|b|e protoco|s or enc|ent h|gh-throughput pur|ncot|on or express|on screen|ng o togged prote|ns hove
become o keg step |n the seorch or drug torgets. ^ two-port studg wos conducted to meosure the robustness o on opt|m|zed,
outomoted protoco| perormed |n o vocuum on o ||qu|d hond||ng stot|on. f|rst, o chessboord studg wos perormed to meosure
poss|b|e cross-contom|not|on. ^ h|st|d|ne-togged prote|n somp|e wos opp||ed to everg second we|| ond the other we||s were
|et emptg. ln the second port, the degree o reproduc|b|||tg wos determ|ned bg the opp||cot|on o s|x d|erent h|st|d|ne-togged
prote|ns rep||coted |n seven rows. The e|uted h|st|d|ne-togged prote|ns were ono|gzed bg $0$-l^CL.
Summary
^utomot|on methods |ncreose throughput ot eoch stoge ond enob|e o h|gh degree o robustness w|th neg||g|b|e cross
contom|not|on ond o h|gh|g cons|stent we||-to-we|| perormonce.
96-well lter plate: |s lu|t|Trop l
Samples: Chessboord studg. Two E. coli somp|es, one
express|ng o |h|st|d|nel
6
-togged recomb|nont prote|n
|l
r
37 000l ond o somp|e where no recomb|nont prote|n
wos expressed.
Reproduc|b|||tg studg. $|x d|erent E. coli somp|es
express|ng d|erent s|zes o recomb|nont prote|ns
|see $0$-l^CL be|owl. Two o the s|x recomb|nont prote|ns
|2 ond 6l were not expressed.
Equilibration buffer: 20 ml Tr|s-C|, 500 ml NoC|,
20 ml |m|dozo|e, p 7.4
Wash buffer: 20 ml Tr|s-C|, 500 ml NoC|,
25 ml |m|dozo|e, p 7.4
Elution buffer: 20 ml Tr|s-C|, 500 ml NoC|,
500 ml |m|dozo|e, p 7.4
Liquid handling station: om||ton llCR0L^3 $T^R
Vacuum station: llCR0L^3 $T^R 3os|c vocuum $gstem |3v$l
100
75
50
37
25
20
15
10

1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6
M M M M
Clarified
bacterial
extracts
Elution,
row 4
Elution,
row 1
Elution,
row 2
Elution,
row 3
Elution,
row 5
Elution,
row 6
Elution,
row 7
(M
r
10
3
)
M B P B P B P B P B P B P B P B P B P B P B P B P B
B : Blank
P : Protein purification
(37 kDa)
B : Blank
P : Protein purification
(37 kDa)
B : Blank
P : Protein purification
(37 kDa)
150
100
75
50
37
25
20
15
10
(M
r
10
3
)
l = lrote|n pur|ncot|on |l
r
37 000l
3 = 3|onk
Acknowledgements: 3. Co||et, l. No|rc|erc-$ovoge ond T. vernet, Ro3|olo|/Loborotorg or locromo|ecu|or Lng|neer|ng, lnst|tut de 3|o|og|e
$tructuro|e CL^-CNR$-UJf, Crenob|e, fronce
H
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23
0pt|m|z|ng pur|ncot|on
cond|t|ons
Products featured: His SpinTrap, ExcelGel SDS Gradient 818, Multiphor II Electrophoresis System,
LMW-SDS Marker Kit
The |m|dozo|e concentrot|on dur|ng b|nd|ng ond wosh|ng |s on |mportont octor thot oects the nno| pur|tg ond g|e|d o the torget
prote|n. Th|s wos demonstroted bg o ser|es o exper|ments |n wh|ch o h|st|d|ne-togged prote|n, ^l3 7-||sl
6
, |l
r
28 000l, wos pur|ned
on |s $p|nTrop us|ng 5, 50, l00, or 200 ml |m|dozo|e concentrot|ons |n the somp|e ond b|nd|ng buers.
97
66
45
30
20
14
(M
r
10
3
)
APB 7-(His)
6
L
M
W

m
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S
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t

m
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,

d
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d

1
:
1
0
5

m
M

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d
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b
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d
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,

d
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1
:
2
5
0

m
M

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d
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g

b
i
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d
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,

d
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1
:
2
1
0
0

m
M

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d
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b
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,

d
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1
:
2
2
0
0

m
M

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d
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g

b
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d
i
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g
,

d
i
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1
:
2
Eluted pool
Column: |s $p|nTrop
Sample: 600 | E.coli 3L-2l |gsote conto|n|ng 400 g
^l3 7-||sl
6
, l
r
28 000
Binding/wash buffer: 20 ml phosphote, 500 ml NoC|,
5 to 200 ml |m|dozo|e, p 7.4
Elution buffer: 20 ml phosphote, 500 ml NoC|,
500 ml |m|dozo|e, p 7.4
Summary
ln th|s opp||cot|on, 50 ml |m|dozo|e prevented the b|nd|ng o most contom|nonts ond |mproved pur|tg. ^dd|ng more |m|dozo|e to
the somp|e ond b|nd|ng buers |ed to o morg|no| |ncreose |n pur|tg but |ower prote|n g|e|d.
SDS-PAGE
24
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lonuo| grov|tg pur|ncot|on
Summary
3oth $0$-l^CL ond vestern b|ot ono|gses o the e|uted roct|ons showed three mojor bonds |nd|cot|ng thot h|st|d|ne togs
were present. The top bond |s the torgeted u||-|ength prote|n ond the bonds be|ow represent truncoted orms o the h|st|d|ne-
togged torget prote|n.
Products featured: His GraviTrap Kit, PhastSystem, PhastGel Gradient 1015, Amersham Hybond ECL,
Anti-His Antibody, ECL Mouse IgG, HRP-linked Whole Ab, Amersham High-Range Rainbow Molecular
Weight Markers
The grov|tg pur|ncot|on method con be eect|ve when gou need to pur|g |orge somp|es |n the obsence o o chromotogrophg
sgstem. 0|rect pur|ncot|on w|thout pr|or c|or|ncot|on o the bocter|o| ce|| |gsotes con be occomp||shed bg us|ng |s Crov|Trop
co|umns. ln th|s exomp|e, o h|gh mo|ecu|or we|ght |h|st|d|nel
l0
-togged prote|n wos pur|ned |n 25 m|n rom 20 m| o c|or|ned E. coli
Jll09 |gsote conto|n|ng TR-l450-||sl
l0
|l
r
~ l30 000l. The e|uted roct|ons were ono|gzed bg $0$-l^CL ond vestern b|ott|ng.
H
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1
:
2
0
F
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,

d
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d

1
:
2
0
E
l
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e

1
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l
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2
N
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c
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(
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s
f
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d

J
M
1
0
9
)

220
97
66
45
20
14
30
(M
r
10
3
)
H
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h

m
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a
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w
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t

m
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t

m
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d
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1
:
2
0
F
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,

d
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d

1
:
2
0
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1
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2
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c
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(
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J
M
1
0
9
)
||sl
l0
-TR-l450
Western blot
Column: |s Crov|Trop
Sample: 20 m| c|or|ned E.coli Jll09 |gsote conto|n|ng ||sl
l0
-TR-l450 |l
r
~l30 000l
Binding/wash buffer: 20 ml phosphote, 500 ml NoC|, 40 ml |m|dozo|e, p 7.4
Elution buffer: 20 ml phosphote, 500 ml NoC|, 500 ml |m|dozo|e, p 7.4
SDS-PAGE
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s
25
lonuo| pur|ncot|on us|ng
o sgr|nge
Products featured: HisTrap FF crude Kit, ExcelGel SDS Gradient 818, Multiphor II Electrophoresis System,
LMW-SDS Marker Kit
0|rect |ood|ng o unc|or|ned ce|| |gsotes decreoses the toto| pur|ncot|on t|me ond |ncreoses the chonces o pur|g|ng sens|t|ve torget
prote|ns w|thout |os|ng oct|v|tg. ln the obsence o o chromotogrophg sgstem, h|st|d|ne-togged prote|ns rom unc|or|ned ce|| |gsotes con
be pur|ned |n o motter o m|nutes us|ng o co|umn ond sgr|nge. ln th|s exomp|e, conven|ent ond s|mp|e pur|ncot|on o o h|st|d|ne-togged
mo|tose b|nd|ng prote|n rom on unc|or|ned somp|e wos perormed us|ng o sgr|nge ond the buers |nc|uded |n |sTrop ff crude K|t.
Summary
l3l-||sl
6
wos qu|ck|g |so|oted rom on unc|or|ned somp|e us|ng o sgr|nge.
0
30 25 20 15
Wash 1
Wash 2
Wash 3
Elution 1
10 5 0
0
0.5
1.0
1.5
2.0
2.5
3.0
100
200
300
400
500
Imidazole
concentration
(mM)
Elution volume (ml)
A
280
Elution 2
Elution 3
(M
r
10
3
)
97
66
45
30
20
14
MBP-(His)
6
L
M
W

m
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:
1
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W
a
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5

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1
0

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1
0

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2
,

3
0

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3
,

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0

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1
,

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0

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2
,

3
0
0

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n

3
,

5
0
0

m
M

i
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a
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Column: |sTrop ff crude l m|
Sample: Unc|or|ned E. coli extroct conto|n|ng
l3l-||sl
6
, l
r
43 000
Flow rate: ^pprox. l m|/m|n
Buffers: lnc|uded |n the |sTrop ff crude K|t
Fraction size: l m|
Equipment: lonuo| pur|ncot|on us|ng o sgr|nge
SDS-PAGE
l m|/roct|on
26
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$|mp|e one-step pur|ncot|on
Summary
L|c|ent pur||cot|on us|ng step e|ut|on wos och|eved |n 35 m|n.
Products featured: HisTrap HP, KTAprime plus
|gh|g pure h|st|d|ne-togged prote|ns con be obto|ned |n o s|ng|e pur|ncot|on step w|th o chromotogrophg sgstem. ln the exomp|e
shown, o step-grod|ent e|ut|on wos used to enc|ent|g pur|g o h|st|d|ne-togged prote|n. The pur|ncot|on process wos s|mp||ned bg
the use o opt|m|zed protoco|s on on ^KT^pr|me p|us.
Sample: C|or|ned homogenote o E. coli express|ng h|st|d|ne-togged prote|n
Column: |sTrop l l m|
Binding buffer: 20 ml phosphote, 0.5 l NoC|, 20 ml |m|dozo|e, p 7.4
Elution buffer: 20 ml phosphote, 0.5 l NoC|, 0.5 l |m|dozo|e, p 7.4
System: ^KT^pr|me p|us
5 10 15 20 25 30 35 min
2.5
2.0
1.5
1.0
0.5
0
100
80
60
40
20
0
A
280
Elution
Buffer %
H
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p
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27
lur|ncot|on o o h|st|d|ne-togged
prote|n expressed |n Pichia pastoris
Products featured: HisTrap FF crude, KTAexplorer, ExcelGel SDS Gradient 818, Multiphor II
Electrophoresis System, LMW-SDS Marker Kit
Lven |n coses where the torget h|st|d|ne-togged prote|n |s expressed ot verg |ow |eve|s, |t |s poss|b|e to obto|n re|ot|ve|g |orge
quont|t|es o pure prote|n becouse the presence o the h|st|d|ne tog eect|ve|g enr|ches the torget prote|n on the onn|tg co|umn.
^n unc|or|ned |gsote o o h|st|d|ne-togged prote|n expressed |n Pichia pastoris wos |ooded d|rect|g onto o co|umn. The prote|n wos
e|uted us|ng o ||neor grod|ent.
Summary
^ h|gh|g pure torget prote|n wos obto|ned rom on unc|or|ned geost |gsote us|ng grod|ent e|ut|on.
Column: |sTrop ff crude 5 m|
Sample: l30 m| o unc|or|ned |gsote o \NR064c |Saccharo-
myces cerevisiae hgdro|osel expressed |n P. pastoris
Binding buffer: 20 ml sod|um phosphote, 500 ml NoC|,
75 ml |m|dozo|e, p 7.4
Elution buffer: 20 ml sod|um phosphote, 500 ml NoC|,
500 ml |m|dozo|e, 3 ml KC|, p 7.4
Flow rate: 5 m|/m|n
System: ^KT^exp|orer l00
mAU
5000
4000
3000
2000
1000
0
100
50
0
0 100 200 300 400 ml
Elution
Buffer %
A
280
(M
r
10
3
)
97
66
45
30
20
14
(His)
6
-YNR064c
L
M
W

m
a
r
k
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s
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t
a
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t

m
a
t
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E
l
u
t
e
d

p
o
o
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,

d
i
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u
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e
d

1
:
2

E
l
u
t
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d

p
o
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,

d
i
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u
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d

1
:
4

SDS-PAGE
28
H
i
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t
i
d
i
n
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a
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g
e
d

p
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i
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s
0ne-step pur|ncot|on comb|ned
w|th on-co|umn reo|d|ng
Products featured: HisTrap HP, KTAexplorer, Biacore System
$ome h|st|d|ne-togged prote|ns ore expressed os |nc|us|on bod|es |n E. coli. lnc|us|on bod|es ore |nso|ub|e oggregotes o m|so|ded
prote|ns |ock|ng b|o|og|co| oct|v|tg. The prob|em con be so|ved bg us|ng o s|mp|e, but enc|ent comb|ned pur|ncot|on ond on-co|umn
reo|d|ng process to produce pure ond oct|ve prote|n. ^ h|st|d|ne-togged scfv 57l ont|bodg rogment expressed os |nc|us|on bod|es
|n E. coli wos so|ub|||zed |n guon|d|ne hgdroch|or|de ond opp||ed to o |sTrop l co|umn. Contom|nonts were removed o||owed bg
on-co|umn reo|d|ng v|o buer exchonge to o nondenotur|ng buer. ^ b|nd|ng ossog between o pept|de der|ved rom the tobocco moso|c
v|rus ond the e|uote |reo|ded h|st|d|ne-togged prote|nl wos perormed us|ng suroce p|osmon resononce |$lRl on o 3|ocore sgstem.
Summary
Us|ng o comb|ned pur||cot|on ond on-co|umn reo|d|ng procedure produced l4% o oct|ve, reo|ded prote|n.
500
400
300
200
100
0
91.5 92 93
pool
94 95 ml 92.5 93.5 94.5
Elution buffer %
100
80
60
40
20
0
500
400
300
200
100
0
0 20 40 60 80 ml
Elution
buffer %
mAU
100
80
60
40
20
0
1
4
2
3 5
Pooled fraction from the purification of refolded
scFv 57P antibody fragment
100
A
280
A
280
mAU
Sample: l0 m| o so|ub|||zed h|st|d|ne-togged s|ng|e cho|n fv ont|bodg rogment |fob 57l
Column: |sTrop l l m|
Solubilizing buffer: 20 ml Tr|s-C|, 6 l Cuo-C|, l ml 0TL, l ml No
2
-L0T^, 0.l ml leob|oc, p 7.5
Denatured binding buffer: 20 ml Tr|s-C|, 5 ml |m|dozo|e, 0.5 l NoC|, 8 l ureo, l ml 0TL, 0.l ml leob|oc, p 7.5
Refolding buffer: 20 ml Tr|s-C|, 5 ml |m|dozo|e, 0.5 l NoC|, 0.5 l org|n|ne-C|, l ml reduced g|utoth|one |C$l, l ml ox|d|zed g|utoth|one |C$$Cl, p 7.5
Native binding buffer: 20 ml Tr|s-C|, l0 ml |m|dozo|e, 0.5 l NoC|, p 7.5
Native elution buffer: 20 ml Tr|s-C|, 500 ml |m|dozo|e, 0.5 l NoC|, p 7.5
Flow rate: l m|/m|n
System: ^KT^exp|orer l0
l. $omp|e opp||cot|on
2. vosh
3. $tort reo|d|ng
4. Lqu|||brot|on o co|umn w|th not|ve b|nd|ng buer
5. $tort e|ut|on
5000
0
10 000
15 000
20 000
-5000
R
e
s
p
o
n
s
e

s
i
g
n
a
l

(
R
U
)
Reference cell
Sample cell
Response difference after subtraction of reference cell
200 400 600 800 1000 1200 1400 1600
s
SPR sensogram
H
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s
29
Summary
The enc|ent, our-step pur|ncot|on protoco| eect|ve|g removed the h|st|d|ne tog rom the prote|n ond g|e|ded ll mg o o h|gh|g
pure ^lCl040.
Unottended our-step pur|ncot|on
ond outomot|c tog removo|
Products featured: HisTrap HP, HiPrep 26/10 Desalting, RESOURCE Q, HiLoad 16/60 Superdex 75 pg,
KTAxpress, ExcelGel SDS Gradient 818, Multiphor II Electrophoresis System, LMW-SDS Marker Kit
^KT^xpress enob|es opt|m|zot|on o c|eovoge cond|t|ons o the h|st|d|ne tog ond outomotes the tosk o mu|t|step pur|ncot|on.
0pt|m|zot|on o ^cTLv proteose c|eovoge cond|t|ons or h|st|d|ne-togged ^lCl040 wos determ|ned us|ng ^KT^xpress. ^ our-step
pur|ncot|on protoco| |nc|ud|ng on-co|umn tog c|eovoge us|ng the opt|m|zed c|eovoge cond|t|ons obto|ned wos then perormed to
och|eve h|gh omounts o pure ^lCl040.
0
500
1000
1500
mAU
1100 1200 1300 1400 1500
B10
11 mg
Cleaved protein
Regeneration
A
280
ml
Elution
buffer %
100
80
60
40
20
0
AC DS IEX GF
97
66
45
30
20
14
(M
r
10
3
)
Cleaved APC1040 (Mr 36 400)
Uncleaved APC1040 (Mr 38 900)
L
M
W

m
a
r
k
e
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s
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t
a
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t

m
a
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i
a
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F
l
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h
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o
u
g
h
P
u
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f
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d
,

c
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a
v
e
d

A
P
C
1
0
4
0

a
f
t
e
r

A
C

D
S

I
E
X

G
F
C
o
n
t
r
o
l
,

u
n
c
l
e
a
v
e
d

A
P
C
1
0
4
0
Sample: ^lCl040-||sl
6
|n E. coli |gsote
Columns: AC: |sTrop l 5 m|
DS: |lrep 26/l0 0eso|t|ng
IEX: RL$0URCL , 6 m|
GF: |Lood l6/60 $uperdex 75 pg
Cleavage conditions: 200 un|ts o ^cTLv proteose/mg prote|n,
8 h |ncubot|on t|me ot room temperoture
AC binding buffer: 50 ml Tr|s-C|, 500 ml NoC|, 20 ml |m|dozo|e, p 7.5
AC cleavage buffer: 50 ml Tr|s-C|, 500 ml NoC|, 50 ml |m|dozo|e, p 7.5
AC elution buffer: 50 ml Tr|s-C|, 500 ml NoC|, 500 ml |m|dozo|e, p 7.5
DS and IEX binding buffer: 50 ml Tr|s-C|, p 8.0
IEX elution buffer: 50 ml Tr|s-C|, l l NoC|, p 8.0
GF buffer: 50 ml Tr|s-C|, l50 ml NoC|, p 7.5
30
H
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d

p
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s
lncreos|ng the pur|ncot|on sco|e
Products featured: HisTrap FF columns, HisPrep FF 16/10, KTAexplorer, ExcelGel SDS Gradient 818,
Multiphor II Electrophoresis System, LMW-SDS Marker Kit
To obto|n sunc|ent moter|o| or chorocter|zot|on stud|es, o sco|e-up exper|ment o o h|st|d|ne-togged mo|tose b|nd|ng prote|n
|l3l-|s
6
l wos perormed. The some prote|n |ood ond ||neor fow rotes were used on o|| three co|umns. Recoverg ond pur|tg o the
e|uted moter|o| wos determ|ned ond compored or o|| three runs.
Summary
The three d|erent sco|e-up pur||cot|ons produced s|m||or resu|ts |n terms o pur|tg ond recoverg.
Columns: |sTrop ff l m|, |sTrop ff 5 m|,
|slrep ff l6/l0 |20 m|l
Sample: l3l-||sl
6
, |n E. coli extroct
Sample volumes: 5.3 m| |l m| co|umnl,
26.5 |5 m| co|umnl, l06 m| |20 m| co|umnl
Binding buffer: 20 ml sod|um phosphote,
25 ml |m|dozo|e, 500 ml NoC|, p 7.4
Elution buffer: 20 ml sod|um phosphote,
500 ml |m|dozo|e, 500 ml NoC|, p 7.4
Flow rates: |sTrop ff l m|. l m|/m|n,
|sTrop ff 5 m|. 5 m|/m|n,
|slrep ff l6/l0. 5 m|/m|n
System: ^KT^exp|orer l00
SDS-PAGE
A
280
0.0 50 100 150 ml
mAU
4000
3500
3000
2500
2000
1500
1000
500
0
33 mg
100
0
50
Elution
buffer %
HisTrap FF 1 ml HisTrap FF 5 ml HisPrep FF 16/10
0.0 5.0 10.0 15.0 20.0 25.0 30.0 40.0 ml
mAU
4000
3500
3000
2500
2000
1500
1000
500
0
6.2 mg
A
280
100
0
50
Elution
buffer %
100
0
50
Elution
buffer %
A
280
mAU
4000
3500
3000
2500
2000
1500
1000
500
0
0 100 200 300 500 400 600 ml
149 mg
700
97
66
45
30
20
14
(M
r
10
3
)
MBP-(His)
6
L
M
W

m
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p
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F
F

1

m
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E
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d

p
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H
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T
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a
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F
F

5

m
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p
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H
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P
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e
p

F
F

1
6
/
1
0
H
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a
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g
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d

p
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s
3l
0rder|ng |normot|on
Product Quantity Code No.
Purication
|sTrop l 5 l m| l7-5247-0l
l00 l m|

l7-5247-05
l 5 m| l7-5248-0l
5 5 m| l7-5248-02
l00 5 m|

l7-5248-05
|s lu|t|Trop l 4 96-we|| n|ter
p|otes
28-4009-89
|s $p|nTrop 50 l00 | 28-40l3-53
|s $p|nTrop K|t 50 x l00 |,
buers
28-932l-7l
|sTrop ff 5 l m| l7-53l9-0l
l00 l m|

l7-53l9-02
5 5 m| l7-5255-0l
l00 5 m|

l7-5255-02
|sTrop ff crude 5 l m| ll-0004-58
l00 l m|

ll-0004-59
5 5 m| l7-5286-0l
l00 5 m|

l7-5286-02
|sTrop ff crude K|t 3 l m|, buers 28-40l4-77
|slrep ff l6/l0 l 20 m| l7-5256-0l
|s lu|t|Trop ff 4 96-we|| n|ter
p|otes
28-4009-90
|s Crov|Trop l0 l m| ll-0033-99
|s Crov|Trop K|t 20 l m|, buers 28-40l3-5l
Detection
^nt|-|s ont|bodg l70 | 27-47l0-0l
LCL louse lgC Rl-||nked
vho|e ^b
l00 | N^93l-l00 |
^mershom gbond LCL
|20 20 cml
l0 sheets RlN20200
^mershom gpern|m LCL
|l8 24 cml
50 sheets 28-9068-36
Related products
|s 3uer K|t l ll-0034-00
N| $ephorose |gh
lerormonce
25 m| l7-5268-0l
l00 m| l7-5268-02
N| $ephorose 6 fost f|ow 5 m| l7-53l8-06
25 m| l7-53l8-0l
l00 m| l7-53l8-02
500 m| l7-53l8-03
|Lood l6/60 $uperdex 30 pg l l20 m| l7-ll39-0l
|Lood 26/60 $uperdex 30 pg l 320 m| l7-ll40-0l
|Lood l6/60 $uperdex 75 pg l l20 m| l7-l068-0l
|Lood 26/60 $uperdex 75 pg l 320 m| l7-l070-0l
|Lood l6/60 $uperdex 200 pg l l20 m| l7-l069-0l
|Lood 26/60 $uperdex 200 pg l 320 m| l7-l07l-0l
|lrep 26/l0 0eso|t|ng l 53 m| l7-5087-0l
4 53 m| l7-5087-02
RL$0URCL 6 m| l l7-ll79-0l
Lxce|Ce| $0$
Crod|ent 8-l8
6 80-l255-53
lhostCe| Crod|ent l0-l5 l0 l7-0540-0l
Llv-$0$ lorker K|t l0 v|o|s l7-0446-0l
^mershom |gh-Ronge
Ro|nbow lo|ecu|or ve|ght
lorkers
250 | RlN756L
Product Quantity Code No.
For more information on Biacore systems and chromatography and/or electrophoresis systems, please visit www.gelifesciences.com

available by specic customer order


32
Strep-tog ll prote|ns
Strep-tog ll |s o smo|| tog o on|g e|ght om|no oc|ds |Trp-$er-|s-lro-C|n-
lhe-C|u-Lgsl w|th o mo|ecu|or we|ght o just l000. The smo|| s|ze o the
tog |s verg benenc|o|, s|nce |n most coses |t does not |nterere w|th structuro|
ond unct|ono| stud|es ond, thereore, does not hove to be removed.
Strep-tog ll b|nds spec|nco||g to Strep-Toct|n ||gond |mmob|||zed on o
$ephorose bose motr|x to g|e|d pure torget prote|n. The b|nd|ng onn|tg
o the Strep-tog ll to the |mmob|||zed ||gond |s neor|g l00-o|d greoter
thon to streptov|d|n, mok|ng $trepToct|n $ephorose |gh lerormonce
su|tob|e or pur|g|ng Strep-tog ll prote|ns. lur|ncot|ons ore run under
phgs|o|og|co| cond|t|ons, ond m||d e|ut|on w|th desth|ob|ot|n preserves
the oct|v|tg o the torget prote|n.
S
t
r
e
p
-
t
a
g

I
I

p
r
o
t
e
i
n
s
33
$|mp|e two-step pur|ncot|on
Products featured: StrepTrap HP, HiLoad 16/60 Superdex 75 pg, KTAprime plus, ExcelGel SDS Gradient
818, Multiphor II Electrophoresis System, LMW-SDS Marker Kit
|gh|g pure Strep-tog ll prote|ns con be obto|ned w|th o s|mp|e chromotogrophg sgstem such os ^KT^pr|me p|us. ||sl
6
-mCherrg-
Strep-tog ll |l
r
~3l 000l wos pur|ned rom on E. coli |gsote us|ng o two-step protoco| |nvo|v|ng onn|tg chromotogrophg w|th o
$trepTrop l l m| co|umn o||owed bg ge| n|trot|on.
AC step:
Column: $trepTrop l l m|
Sample: l0 m| o E. coli c|or|ned |gsote
conto|n|ng ||sl
6
-mCherrg-Strep-tog ll
Flow rate: l m|/m|n
Binding buffer: l00 ml Tr|s-C|, l50 ml NoC|,
l ml L0T^, p 8.0
Elution buffer: 2.5 ml desth|ob|ot|n |n
b|nd|ng buer
GF step:
Column: |Lood l6/60 $uperdex 75 pg
Sample: L|uted poo| |2 m|l rom $trepTrop l l m|
Flow rate: l m|/m|n
Buffer: l3$ buer, p 7.4
System: ^KT^pr|me p|us
2000
1500
0
A
280
mAU
1000
500
0 10 20 30 40 ml
40
50
30
60
70
0
20
10
0 10 20 30 40 50 60 ml
A
280
mAU
Summary
|gh|g pure Strep-tog ll recomb|nont prote|n wos obto|ned w|th th|s s|mp|e two-step pur||cot|on protoco|. The two
contom|nonts o obout l
r
l0 000 ond 2l 000, respect|ve|g mog be coused bg rogmentot|on o the torget prote|n dur|ng
$0$-l^CL ono|gs|s.
(Histidine)
6
tag Strep-tag II mCherry Full-length target protein
(Histidine)
6
tag
Strep-tag II
mCherry fragment
mCherry fragment
M
r
= 30 000
PreScission MYG (85-87) TEV
PreScission
MYG (85-87) TEV
M
r
9558
M
r
20 741
L
M
W

m
c
r
k
e
r
s
N
e
g
c
t
i
v
e

c
o
n
t
r
o
l
S
c
m
p
l
e
,

d
i
l
u
t
e
d

1
:
1
5
f
l
o
w
t
h
r
o
u
g
h

l
u
t
e
d

p
o
o
l

l
u
t
e
d

p
o
o
l
,

d
i
l
u
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d

1
:
5

l
u
t
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d

p
o
o
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,

d
i
l
u
t
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d

1
:
5
Strep1rcp HP
luted frcctions,
HiLocd 16l60
Superdex 75 pg
97
66
45
30
20
14
(M
r
10
3
)
SDS-PAGE
AC step GF step
34
S
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p
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I
I

p
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o
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e
i
n
s
Unottended pur|ncot|on o o
prote|n expressed |n |nsect ce||s
Products featured: StrepTrap HP, KTAxpress
||sl
6
-Strep-tog ll-prote|n |l
r
l5 400l wos expressed |n |nsect ce||s rom o 3ocu|ov|rus express|on vector ond pur|ned. The h|gh
spec|nc|tg o the Strep-tog ll to the Strep-Toct|n ||gond wos ut|||zed |n o one-step pur|ncot|on procedure us|ng $trepTrop l co|umn
to obto|n h|gh|g pure torget prote|n.
Column: $trepTrop l 5 m|
Sample: l75 m| o |nsect ce|| |gsote conto|n|ng
||sl
6
-Strep-tog ll-prote|n |l
r
~l5 400l
Binding buffer: l00 ml Tr|s-C|, l50 ml NoC|, l ml L0T^, p 8.0
Elution buffer: 2.5 ml desth|ob|ot|n |n b|nd|ng buer
Flow rates: 5 m|/m|n |l m|/m|n dur|ng somp|e opp||cot|onl
System: ^KT^xpress
188
98
62
49
38
28
17
14
60
30
(M
r
10
3
)
(His)
6
-Strep-
tag II-protein
M
a
r
k
e
r
s
L
y
s
a
t
e
F
l
o
w
t
h
r
o
u
g
h
E
l
u
t
e
d

t
a
r
g
e
t

p
r
o
t
e
i
n

p
o
o
l
Summary
Th|s s|mp|e, s|ng|e-step process produced 3.7 mg o h|gh|g pure prote|n.
SDS-PAGE
Acknowledgements: l. N||sson, R. $vensson ond L. o|mgren, 3|ov|trum, $tockho|m, $weden
35
S
t
r
e
p
-
t
a
g

I
I

p
r
o
t
e
i
n
s
lncreos|ng the pur|ncot|on sco|e
Products featured: StrepTrap HP columns, XK 26/20 column, StrepTactin Sepharose High Performance,
KTAexplorer
$co|e-up o prote|ns con be och|eved bg |ncreos|ng the bed vo|ume wh||e keep|ng the res|dence t|me constont. Th|s opprooch
mo|nto|ns chromotogroph|c perormonce dur|ng sco|e-up. The prote|n used wos o fuorescent prote|n, ||sl
6
-mCherrg-Strep-tog ll, |n
E. coli |gsote, wh|ch con be detected ot 587 nm os we|| os 280 nm. lur|ncot|on on o $trepTrop l l m| co|umn wos nrst perormed
ond then sco|ed up to the 5 m| co|umn o||owed bg urther sco|e-up to o 29 m| K 26/20 co|umn pocked w|th $trepToct|n $ephorose
|gh lerormonce. The prote|n |ood wos |ncreosed nve-o|d |n eoch sco|e-up step.
Summary
The co|umns gove comporob|e resu|ts, con|rm|ng the eose ond reproduc|b|||tg o sco||ng up the pur||cot|on rom $trepTrop l
co|umns to o |orger K 26/20 co|umn pocked w|th $trepToct|n $ephorose |gh lerormonce.
0
1000
2000
3000
A280
mAU
0
1000
2000
3000
A587
mAU
0.0 10.0 20.0 ml
= A280
= A587
= Elution buffer, %B
Columns: $trepTrop l l m|, $trepTrop l 5 m|, $trepToct|n $ephorose
|gh lerormonce pocked |n K 26/20, 29 m|, bed he|ght 5.5 cm
Sample: ||sl
6
-mCherrg-Strep-tog ll |l
r
~3l 000l, |n E. coli |gsote
Sample volumes: 4.2 m| |$trepTrop l l m|l, 2l m| |$trepTrop l 5 m|l,
l05 m| |K 26/20 co|umnl
Regeneration: 3 co|umn vo|umes |Cvl d|st|||ed woter, 3 Cv 0.5 l No0,
3 Cv d|st|||ed woter
Binding buffer: l00 ml Tr|s-C|, l50 ml NoC|, l ml L0T^, p 8.0
Elution buffer: 2.5 ml desth|ob|ot|n |n b|nd|ng buer
Flow rates: $trepTrop l l m|. l.0 m|/m|n |0.5 m|/m|n dur|ng somp|e
|ood|ng ond regenerot|on w|th 0.5 l No0l.
$trepTrop l 5 m|. 5.0 m|/m|n |2.5 m|/m|n dur|ng somp|e |ood|ng ond
regenerot|on w|th 0.5 l No0l.
K 26/20 co|umn. l3 m|/m|n |6.5 m|/m|n dur|ng regenerot|on w|th
0.5 l No0l
System: ^KT^exp|orer
A280
mAU
A587
mAU
0
1000
2000
3000
0
1000
2000
3000
0 50 100 150 ml
A280
mAU
A587
mAU
0
1000
2000
3000
0
1000
2000
3000
0 200 400 600 700 ml
StrepTrap HP 1 ml StrepTrap HP 5 ml StrepTactin Sepharose
High Performance XK 26/20
36
S
t
r
e
p
-
t
a
g

I
I

p
r
o
t
e
i
n
s
0rder|ng |normot|on
Purication
$trepTrop l 5 l m| 28-9075-46
l 5 m| 28-9075-47
5 5 m| 28-9075-48
$trepToct|n $ephorose |gh
lerormonce
l0 m| 28-9355-99
50 m| 28-9356-00
Related products
|Lood l6/60 $uperdex 30 pg l l20 m| l7-ll39-0l
|Lood 26/60 $uperdex 30 pg l 320 m| l7-ll40-0l
|Lood l6/60 $uperdex 75 pg l l20 m| l7-l068-0l
|Lood 26/60 $uperdex 75 pg l 320 m| l7-l070-0l
|Lood l6/60 $uperdex 200 pg l l20 m| l7-l069-0l
|Lood 26/60 $uperdex 200 pg l 320 m| l7-l07l-0l
|lrep 26/l0 0eso|t|ng l 53 m| l7-5087-0l
4 53 m| l7-5087-02
Lxce|Ce| $0$ Crod|ent 8-l8 6 80-l255-53
Llv-$0$ lorker K|t l0 v|o|s l7-0446-0l
Product Quantity Code No.
For more information on chromatography and/or electrophoresis systems, please visit www.gelifesciences.com
37
38
l3l-togged prote|ns
lo|tose b|nd|ng prote|n |l3ll |s o useu| onn|tg tog thot con |ncreose the
express|on |eve| ond so|ub|||tg o the resu|t|ng togged prote|n. The l3l tog
o|so promotes proper o|d|ng o the ottoched prote|n. $|nce l3l |ncreoses
so|ub|||tg, the tog |s port|cu|or|g useu| or recomb|nont prote|ns thot
occumu|ote |n on |nso|ub|e orm ||nc|us|on bod|esl.
^nn|tg pur|ncot|on tokes p|oce under phgs|o|og|co| cond|t|ons ond m||d
e|ut|on |s perormed us|ng mo|tose. The m||d e|ut|on preserves the oct|v|tg
o the l3l-togged prote|n.
39
M
B
P
-
t
a
g
g
e
d

p
r
o
t
e
i
n
s
$|mp|e two-step pur|ncot|on
Products featured: MBPTrap HP, HiLoad 16/60 Superdex 200 pg, KTAprime plus, ExcelGel SDS Gradient 818,
Multiphor II Electrophoresis System, LMW-SDS Marker Kit
l3l2-poromgos|n--$o| wos pur|ned |n two steps w|th on l3lTrop l l m| co|umn, o||owed bg ge| n|trot|on. The pur|ncot|on
process wos s|mp||ned w|th preprogrommed methods ond opt|m|zed protoco|s on on ^KT^pr|me p|us.
Summary
lnc|us|on o the second ge| ||trot|on step eect|ve|g removed |mpur|t|es ond contom|nonts to produce o pure torget prote|n.
2000
1500
0
1000
500
0 10 20 30 40 ml
A
280
mAU
100
120
50
A
280
mAU
0
0 20 40 60 80 100 120 ml
AC step:
Column: l3lTrop l l m|
Sample: 4 m| l3l2-poromgos|n--$o| |n c|or|ned E. coli |gsote
Flow rate: l m|/m|n
Binding buffer: 20 ml Tr|s-C|, 200 ml NoC|, l ml L0T^,
l ml 0TT, p 7.4
Elution buffer: l0 ml mo|tose |n b|nd|ng buer
GF step:
Column: |Lood l6/60 $uperdex 200 pg
Sample: L|uted poo| |2 m|l rom l3lTrop l l m|
Flow rate: l m|/m|n
Buffer: l3$ buer, p 7.4
97
66
45
30
20
14
(M
r
10
3
)
MBP*2-
paramyosin-
-Sal
L
M
W

m
a
r
k
e
r
s
S
a
m
p
l
e
,

d
i
l
u
t
e
d

1
:
1
5
F
l
o
w
t
h
r
o
u
g
h
,

d
i
l
u
t
e
d

1
:
1
5
,

M
B
P
T
r
a
p

H
P
M
B
P
T
r
a
p

H
P
H
i
L
o
a
d

S
u
p
e
r
d
e
x

1
6
/
6
0

2
0
0

p
g
Eluted pool
SDS-PAGE
40
M
B
P
-
t
a
g
g
e
d

p
r
o
t
e
i
n
s
Lnc|ent two-step pur|ncot|on o o
prote|n |nvo|ved |n metobo||c d|seose
Products featured: MBPTrap HP, Superdex 200 prep grade, XK 16/20, KTAprime
lC^0 |l
r
85 500l |s o homotetromer prote|n |nvo|ved |n metobo||c d|seose. ln th|s studg, lC^0 wos pur|ned or stob|||tg o|d|ng ond
k|net|c stud|es. The pur|tg o the e|uted roct|ons wos determ|ned bg $0$-l^CL ono|gs|s. $ome truncoted orms o the torget prote|n,
os we|| os prote|n oggregotes were detected rom the nrst step ond were eect|ve|g removed |n the second step.
Summary
The torget prote|n wos h|gh|g concentroted ond |t e|uted |n o smo|| vo|ume rom the |rst o|n|tg step. Th|s removes the need to
concentrote the prote|n pr|or to ge| ||trot|on thus sov|ng t|me, cost, ond prote|n somp|e.
1000
1500
2000
2500
500
A280
mAU
0
0 20 40 60 80 100 ml 120
200
300
400
100
0
0 20 40 60 80 ml
A280
mAU
170
130
95
72
56
43
34
26
11
17
(M
r
10
3
)
MBP-
MCAD
M
o
l
e
c
u
l
a
r

w
e
i
g
h
t

m
a
r
k
e
r
s
S
t
a
r
t

m
a
t
e
r
i
a
l
,

d
i
l
u
t
e
d

1
:
6
F
l
o
w
t
h
r
o
u
g
h

M
B
P
T
r
a
p

H
P
,

d
i
l
u
t
e
d

1
:
6
E
l
u
t
e
d

f
r
a
c
t
i
o
n
s

f
r
o
m

M
B
P
T
r
a
p

H
P
E
l
u
t
e
d

f
r
a
c
t
i
o
n
s

f
r
o
m

S
u
p
e
r
d
e
x

2
0
0

p
g
AC step
Column: l3lTrop l 5 m|
Sample: l5 m| o N-term|no| l3l-lC^0 |n E. coli |gsote
Binding buffer: 20 ml Tr|s-C|, 200 ml NoC|, l ml L0T^,
l ml 0TT, p 7.4
Elution buffer: l0 ml mo|tose |n b|nd|ng buer
Flow rate: 5.0 m|/m|n |0.5 m|/m|n dur|ng somp|e |ood|ngl
System: ^KT^pr|me
GF step
Column: $uperdex 200 pg |n K l6/20
Sample: 2 m| o e|uted roct|on rom l3lTrop l 5 m|
Buffer: 20 ml LlL$, 200 ml NoC|, p 7.0
Flow rate: 0.4 m|/m|n
System: ^KT^pr|me
SDS-PAGE
Acknowledgements: L. l. lo|er, 0r. von ounersches K|ndersp|to|, lun|ch, Cermong
4l
M
B
P
-
t
a
g
g
e
d

p
r
o
t
e
i
n
s
Unottended outomoted
two-step pur|ncot|on
Products featured: MBPTrap HP, HiLoad 16/60 Superdex 200 pg, KTAxpress
l3l-togged opopt|n prote|n |l
r
~ 60 000l wos pur|ned |n o two-step procedure us|ng ^KT^xpress w|th onn|tg chromotogrophg |^Cl
ond ge| n|trot|on |Cfl protoco|s. The prote|n wos |ntended or crgsto|||zot|on screen|ng ond unct|ono| stud|es. The e|uted peok rom
the l3lTrop co|umn wos outomot|co||g co||ected |n o |oop ond |njected onto the ge| n|trot|on co|umn.
Summary
The use o on|g two steps |n on outomoted protoco| w|th ^KT^xpress |ncreoses occurocg, o||ows or honds-o operot|on, ond
e||m|notes humon error.
AC column: l3lTrop l 5 m|
Sample: l5 m| o l3l-opopt|n |n E. coli |gsote, l
r
~60 000
Flow rate: 5 m|/m|n
Binding buffer: 20 ml Tr|s-C|, 200 ml NoC|, l ml L0T^,
l ml 0TT, p 7.4
Elution buffer: l0 ml mo|tose |n b|nd|ng buer
GF column: |Lood l6/60 $uperdex 200 pg
Sample: Co||ected poo| rom l3lTrop l
Flow rate: 0.3 m|/m|n
Buffer: l0 ml sod|um phosphote, l40 ml NoC|, 0.5 l L0T^, p 7.2
System: ^KT^xpress
Elution buffer
%B
0
500
1000
1500
2000
A
280
mAU
0
20
40
60
80
100
60 80 100 120 140 160 180 ml
AC GF
SDS-PAGE
Acknowledgements: R. |mmermon, Le|den Un|vers|tg, 2333CC Le|den, The Nether|onds
250
130
100
70
55
35
27
15
10
(M
r
10
3
)
M
o
l
e
c
u
l
a
r

w
e
i
g
h
t

m
a
r
k
e
r
Eluted fractions from gel filtration
42
M
B
P
-
t
a
g
g
e
d

p
r
o
t
e
i
n
s
lncreos|ng the pur|ncot|on sco|e
Products featured: MBPTrap HP columns, XK 26/20 column, Dextrin Sepharose High Performance,
KTAexplorer, ExcelGel SDS Gradient 818, Multiphor II Electrophoresis System, LMW-SDS Marker Kit
To |nvest|gote the reproduc|b|||tg o sco|e-up us|ng l3lTrop co|umns, l3l2--go|octos|dose |l
r
~l58 000l, o recomb|nont togged
mu|t|mer, wos pur|ned on l3lTrop l l m| ond 5 m| co|umns, wh|ch ore prepocked w|th 0extr|n $ephorose |gh lerormonce
med|um. further sco|e-up wos perormed on on K 26/20 co|umn, pocked w|th the some chromotogrophg med|um. $omp|e |ood
wos |ncreosed nve-o|d or eoch sco|e-up step.
Summary
The co|umns gove comporob|e resu|ts w|th h|gh pur|tg ond s|m||or g|e|ds |obout 60%, doto not shownl, con|rm|ng the eose
ond reproduc|b|||tg o sco||ng up pur||cot|ons rom l3lTrop l co|umns to on K 26/20 co|umn pocked w|th the some
chromotogrophg med|um.
Columns: l3lTrop l l m|, l3lTrop l 5 m|, 0extr|n $ephorose |gh
lerormonce pocked |n K 26/20, 29 m|, bed he|ght 5.5 cm
Sample: l3l2--go|octos|dose |l
r
~l58 000l |n E. coli |gsote
Sample volumes: 5 m| |l3lTrop l l m|l, 25 m| |l3lTrop l 5 m|l,
l25 m| |K 26/20 co|umnl
Binding buffer: 20 ml Tr|s-C|, 200 ml NoC|, l ml L0T^, l ml 0TT, p 7.4
Elution buffer: l0 ml mo|tose |n b|nd|ng buer
Flow rates: l3lTrop l l m|. l.0 m|/m|n |0.5 m|/m|n dur|ng somp|e |ood|ngl
l3lTrop l 5 m|. 5.0 m|/m|n |2.5 m|/m|n dur|ng somp|e |ood|ngl
K 26/20 co|umn. l3 m|/m|n
System: ^KT^exp|orer
0
500
1000
1500
2000
2500
3000
A
280
mAU
0.0 5.0 10.0 15.0 20.0 ml
80
100
Elution Buffer %
60
40
20
0
0
500
1000
1500
2000
2500
3000
0 100 200 300 400 500 ml
80
100
60
40
20
0
600
A
280
mAU
Elution Buffer %
97
66
45
30
20
14
(M
r
10
3
)
MBP2*--
galactosidase
L
M
W

m
a
r
k
e
r
s
M
B
P
T
r
a
p

H
P

1

m
l
,

d
i
l
u
t
e
d

1
:
6
M
B
P
T
r
a
p

H
P

5

m
l
,

d
i
l
u
t
e
d

1
:
1
2
X
K

2
6
/
2
0
,

d
i
l
u
t
e
d

1
:
1
2
M
B
P
T
r
a
p

H
P

1

m
l
,

d
i
l
u
t
e
d

1
:
3
M
B
P
T
r
a
p

H
P

5

m
l
,

d
i
l
u
t
e
d

1
:
3
X
K

2
6
/
2
0
,

d
i
l
u
t
e
d

1
:
3
S
t
a
r
t

m
a
t
e
r
i
a
l
,

d
i
l
u
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1
:
3
Flowthrough Eluted pool
MBPTrap HP 1 ml Dextrin Sepharose High Performance XK 26/20
SDS-PAGE
43
M
B
P
-
t
a
g
g
e
d

p
r
o
t
e
i
n
s
0rder|ng |normot|on
Purication
l3lTrop l 5 l m| 28-9l87-78
l 5 m| 28-9l87-79
5 5 m| 28-9l87-80
0extr|n $ephorose |gh
lerormonce
25 m| 28-9355-97
l00 m| 28-9355-98
Purication
|Lood l6/60 $uperdex 30 pg l l20 m| l7-ll39-0l
|Lood 26/60 $uperdex 30 pg l 320 m| l7-ll40-0l
|Lood l6/60 $uperdex 75 pg l l20 m| l7-l068-0l
|Lood 26/60 $uperdex 75 pg l 320 m| l7-l070-0l
|Lood l6/60 $uperdex 200 pg l l20 m| l7-l069-0l
|Lood 26/60 $uperdex 200 pg l 320 m| l7-l07l-0l
Lxce|Ce| $0$ Crod|ent 8-l8 6 80-l255-53
Llv-$0$ lorker K|t l0 v|o|s l7-0446-0l
$uperdex 200 prep grode l50 m| l7-l043-0l
K l6/20 emptg co|umn l l8-8773-0l
Product Quantity Code No.
For more information on Biacore systems and chromatography and/or electrophoresis systems, please visit www.gelifesciences.com
44
0uo|-togged prote|ns
Recomb|nont prote|ns con be des|gned to conto|n N- or C-term|no| onn|tg
togs. The |nc|us|on o two d|erent togs |n o construct resu|ts |n o torget
prote|n thot con be pur|ned w|th d|erent onn|tg chromotogrophg med|o
thus produc|ng o purer prote|n w|th o stro|ghtorword pur|ncot|on scheme.
l d|erent togs ore pos|t|oned on eoch end o the prote|n, o duo|-togged
pur|ncot|on scheme wou|d normo||g produce o u||-|ength prote|n. The
onn|tg tog con be c|eoved | c|eovoge s|tes ore |nc|uded between the tog
ond the torget prote|n.
45
D
u
a
l
-
t
a
g
g
e
d

p
r
o
t
e
i
n
s
0ne versus two onn|tg steps
Products featured: HisTrap HP, GSTrap 4B, KTAxpress, ExcelGel SDS Gradient 818, Multiphor II
Electrophoresis System, LMW-SDS Marker Kit
To compore pur|ncot|on us|ng e|ther one tog or o comb|not|on o two d|erent togs, o duo|-togged green fuorescent prote|n
||s
6
-Cfl-C$Tl wos pur|ned |n three d|erent wogs. lur|ncot|on on |sTrop l showed thot the med|um hod onn|tg or other
not|ve prote|ns w|th exposed h|st|d|ne res|dues os we||, whereos ut|||z|ng the h|gh spec|nc|tg o the C$T tog or the g|utoth|one
||gond on C$Trop 43 gove o much purer prote|n. The comb|not|on o the two togs produced the h|ghest pur|tg.
Summary
0uo|-togged pur||cot|on coners o sgnerg|st|c bene|t |h|gher pur|tgl thot somet|mes e|udes s|ng|e-togged pur||cot|on schemes.
Sample: ||sl
6
-Cfl-C$T |n E.coli |gsote
Columns:
Afnity chromatography: |sTrop l 5 m| ond C$Trop 43 5 m|
Desalting: |lrep 26/l0 0eso|t|ng
Buffers:
HisTrap HP
Binding buffer: 20 ml l3$, 20 ml |m|dozo|, 0.5 l NoC|, p 7.4
Elution buffer: 20 ml l3$, 500 ml |m|dozo|, 0.5 l NoC|, p 7.4
GSTrap 4B:
Binding buffer: l0 ml l3$, p 7.4
Elution buffer: 50 ml Tr|s, 20 ml reduced g|utothone, p 8
HiPrep 26/10 Desalting:
Buffer: 50 ml Tr|s-C| , l50 ml NoC|, p 7.5
HiPrep 16/10
HiPrep 16/10
HiPrep 16/10
HiPrep 16/10
Run 1 Run 2 Run 3
HisTrap HP HiPrep 26/10
Desalting
GSTrap 4B HiPrep 26/10
Desalting
GSTrap 4B HiPrep 26/10
Desalting
HisTrap HP HiPrep 26/10
Desalting
96
66
45
30
20
14
(M
r
10
3
)
L
M
W

m
a
r
k
e
r
s
F
l
o
w
t
h
r
o
u
g
h

r
u
n

1
E
l
u
t
i
o
n

r
u
n

1
F
l
o
w
t
h
r
o
u
g
h

r
u
n

2
E
l
u
t
i
o
n

r
u
n

2
F
l
o
w
t
h
r
o
u
g
h

r
u
n

3
E
l
u
t
i
o
n

r
u
n

3
(His)
6
-GFP-GST
SDS-PAGE
D
u
a
l
-
t
a
g
g
e
d

p
r
o
t
e
i
n
s
46
lncreosed pur|tg w|th
duo|-togged expressed prote|n
Products featured: HisTrap HP, StrepTrap HP, KTAxpress
$|nce o h|gh degree o pur|tg |s cruc|o| or successu| unct|ono| stud|es, pur|tg resu|ts o the two-step method were compored w|th
those rom s|ng|e-step pur|ncot|ons. ^ duo|-togged Strep tog ll-|h|st|d|nel
6
prote|n |l
r
~l5 400l expressed |n E. coli wos pur|ned or
method deve|opment us|ng o two-step procedure compr|s|ng |mmob|||zed meto| onn|tg chromotogrophg |ll^Cl o||owed bg onn|tg
chromotogrophg on o $trepTrop l co|umn. ^|| the exper|ments were conducted ot 4C to preserve prote|n stob|||tg.
Summary
$0$-l^CL resu|ts c|eor|g demonstrote the bene|ts o o duo|-togged opprooch to prote|n pur||cot|on, espec|o||g when h|gh
pur|tg |s requ|red.
188
98
62
49
38
28
(M
r
10
3
)
17
14
6
3
Dual-tagged
Strep-tag II-(His)
6
M
o
l
e
c
u
l
a
r

w
e
i
g
h
t

m
a
r
k
e
r
F
l
o
w
t
h
r
o
u
g
h
E
l
u
t
e
d

p
o
o
l
F
l
o
w
t
h
r
o
u
g
h
E
l
u
t
e
d

p
o
o
l
E
l
u
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Individual
HisTrap HP
HisTrap HP +
StrepTrap HP
Individual
StrepTrap HP
Sample: l5 m| Strep-tog ll-|h|st|d|nel
6
prote|n
|l
r
~l5 400l |n E. coli |gsote
Individual HisTrap HP purication
Column: |sTrop l l m|
Binding buffer: 20 ml sod|um phosphote,
500 ml NoC|, 20 ml |m|dozo|e, p 7.5
Elution buffer: 20 ml sod|um phosphote,
500 ml NoC|, 500 ml |m|dozo|e, p 7.5
Flow rate: 0.8 m|/m|n
Individual StrepTrap HP purication
Column: $trepTrop l l m|
Binding buffer: l00 ml Tr|s-C|, l50 ml NoC|,
l ml L0T^, p 8.0
Elution buffer: 2.5 ml desth|ob|ot|n |n
b|nd|ng buer
Flow rate: 0.8 m|/m|n
Two-step HisTrap HP and StrepTrap HP purication
Column: |sTrop l l m|
Binding buffer: 20 ml sod|um phosphote,
500 ml NoC|, 20 ml |m|dozo|e, p 7.5
Elution buffer: 20 ml sod|um phosphote,
500 ml NoC|, 500 ml |m|dozo|e, p 7.5
Flow rate: 0.8 m|/m|n
Column: $trepTrop l l m|
Sample: L|uted roct|on rom |sTrop l, l m|
Binding buffer: l00 ml Tr|s-C|, l50 ml NoC|, l ml
L0T^, p 8.0
Elution buffer: 2.5 ml desth|ob|ot|n |n b|nd|ng buer
Flow rate: 0.2 m|/m|n
System: ^KT^xpress
SDS-PAGE
Acknowledgements: l. N||sson, R. $vensson ond L. o|mgren, 3|ov|trum, $tockho|m, $weden
47
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p
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lmpoct o revers|ng the order
o onn|tg pur|ncot|on
Products featured: StrepTrap HP, HisTrap HP, KTAxpress, ExcelGel SDS Gradient 818, Multiphor II
Electrophoresis System, LMW-SDS Marker Kit
To evo|uote whether the order o onn|tg co|umns |n o chosen pur|ncot|on method motters w|th respect to g|e|d ond pur|tg. ||sl
6
-
mCherrg-Strep-tog ll duo|-togged torget prote|n, wos pur|ned us|ng $trepTrop l o||owed bg |sTrop l ond subsequent|g |n the
reverse order. $0$-l^CL ono|gs|s showed thot the pur|tg o the prote|n wos the some, |rrespect|ve o the order o onn|tg pur|ncot|on
used. ^n N-term|no||g truncoted torget prote|n |dent|ned bg moss spectrometrg |doto not shownl, however, possed through the
|sTrop l co|umn more s|ow|g thon nontogged prote|ns, wh|ch occounts or the two peoks observed |n the fowthrough.
Columns: $trepTrop l l m|, |sTrop l l m|
Sample: ||sl
6
-mCherrg-Strep-tog ll |n E. coli |gsote
Binding buffer (StrepTrap HP): l00 ml Tr|s-C|, l50 ml NoC|,
l ml L0T^, p 8.0
Elution buffer (StrepTrap HP): l00 ml Tr|s-C|, l50 ml NoC|,
l ml L0T^, 2.5 ml desth|ob|ot|n, p 8.0
Binding buffer (HisTrap HP): 20 ml phosphote, 500 ml NoC|,
5 ml lm|dozo|e, p 7.4
Elution buffer (HisTrap HP): 20 ml phosphote, 500 ml NoC|,
500 ml lm|dozo|e, p 7.4
System: ^KT^xpress
Summary
The some omount |0.9 mgl o prote|n wos produced |n both exper|ments ond the pur|tg o the torget prote|n wos not oected
bg the order o the o|n|tg co|umns used.
0
100
200
300
400
500
600
700
mAU
70 80 90 100 110 ml
A
280
StrepTrap HP
elution
HisTrap HP
elution
HisTrap HP
flow through
66
30
M
r
10
3
96
45
20.1
14.4
(His)
6
-mCherry-Strep-tag II
0
500
1000
1500
2000
mAU
70 80 90 100 110 120 130 140 ml
A
280
HisTrap HP
elution
StrepTrap HP
elution
Loop
wash
StrepTrap HP
flow through
M
r
10
3
96
45
20.1
14.4
66
30
(His)
6
-mCherry-Strep-tag II
for contoct |normot|on or gour |oco| once,
p|eose v|s|t. www.ge||esc|ences.com/contoct
CL eo|thcore 3|o-$c|ences ^3
3jorkgoton 30
75l 84 Uppso|o
$weden
www.ge||esc|ences.com
imagination at work
CL, |mog|not|on ot work ond CL monogrom ore trodemorks o
Cenero| L|ectr|c Compong.
^KT^des|gn, ^KT^exp|orer, ^KT^pr|me, ^KT^xpress, ^mershom,
3|ocore, LCL, Lxce|Ce|, Crov|Trop, C$Tprep, C$Trop, |Lood,
|sTrop, |slrep, |lrep, gbond, gpern|m, l3lTrop,
lu|t|Trop, lhostCe|, lre$c|ss|on, RL$0URCL, $ephorose,
$uperdex, $p|nTrop, $trepTrop, ond UNlC0RN ore trodemorks
o CL eo|thcore compon|es.
|st|d|ne-togged prote|ns. lur|ncot|on ond preporot|on o
us|on prote|ns ond onn|tg pept|des compr|s|ng ot |eost two
odjocent h|st|d|ne res|dues mog requ|re o ||cense under U$
potent numbers 5,284,933 ond 5,3l0,663, ond equ|vo|ent
potents ond potent opp||cot|ons |n other countr|es |oss|gnee.
omon Lo Roche, lncl.
N| $ephorose 6 fost f|ow. Th|s products |s covered bg U$ pot
No 6 623 655 ond the|r equ|vo|ents |n other countr|es.
pCL vectors ore to be used or sc|ent|nc |nvest|got|on
ond reseorch ond or no other purpose whotsoever ond o
||cense or commerc|o| use o the ||censed products ond the
processes c|o|med |n U$ potent 5,654,l76 ond equ|vo|ent
potents ond potent opp||cot|ons |n other countr|es must be
negot|oted d|rect|g w|th l||||pore Corp |ormer|g Chem|con
lnternot|ono| lncl bg the purchoser pr|or to such use.
$trepTrop l ond $trepToct|n $ephorose |gh lerormonce
These products ore covered bg U$ potent number 6,l03,493
ond equ|vo|ent potents ond potent opp||cot|ons |n other
countr|es. The purchose o $trepTrop l ond $trepToct|n
$ephorose |gh lerormonce |nc|udes o ||cense under such
potents or non-pront ond |n-house reseorch on|g. l|eose
contoct l3^ ||no|bo-go.coml or urther |normot|on on
||censes or commerc|o| use o $trepToct|n.
^|| th|rd portg trodemorks ore the propertg o the|r respect|ve
owners.
2008 Cenero| L|ectr|c Compong-^|| r|ghts reserved.
f|rst pub||shed ^ugust 2008
^|| goods ond serv|ces ore so|d subject to the terms ond
cond|t|ons o so|e o the compong w|th|n CL eo|thcore
wh|ch supp||es them. ^ copg o these terms ond cond|t|ons
|s ovo||ob|e on request. Contoct gour |oco| CL eo|thcore
representot|ve or the most current |normot|on.
CL eo|thcore 3|o-$c|ences ^3
3jorkgoton 30
75l 84 Uppso|o
$weden
CL eo|thcore Lurope, Cmb
lunz|nger $trosse 5
0-79lll fre|burg
Cermong
CL eo|thcore 3|o-$c|ences Corp.
800 Centenn|o| ^venue, l.0. 3ox l327
l|scotowog, NJ 08855-l327
U$^
CL eo|thcore 3|o-$c|ences KK
$onken 3|dg., 3-25-l, gokun|ncho
$h|njuku-ku, Tokgo l69-0073
Jopon
28-9353-64 ^^ 08/2008


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