BO CO THC TP CC K THUT C BN TRONG CNG NGH SINH HC Ging vin hng dn: ThS: Nguyn Th Hng Loan Sinh

vin: Giang Huy Dim I. Cu hi u bi: 1. Ti sao phi tinh sch protein?

Tr li: V khi protein dng tinh sch s c hot tnh mnh hn dng th.Hn na, mun nghin cu cu trc ca protein no th cn phi c dng tinh khit ca n. 2. C nhng cch no tinh sch protein Tr li: C th tinh sch bng in di khng bin tnh, cc phng php sc k, ly tm siu tc,... Nhng in di t dng v in di ch tinh sch c 1 lng nh. 3. Sau khi tinh sch, lm th no thu nhn protein mong mun? Tr li: Ta c th dng phng php in di xem c ln bng chun khng. Phng php khi ph s gip kim tra c on peptit m ta xc nh trc hay khng. Hay mt phng php khc l min dch hunh quang. Lu : d dng phng php no th trc khi thu nhn protein u phi kim tr hot tnh protein xem c ng protein khng?

II. Sc k trao i ion - IEC 1. Gii thiu chung - Tng tc ion l c s ca vic tch v tinh ch protein bng sc k trao i ion (IEC) - IEC hin ti l k thut tch protein ph bin nht, c dng trong cc quy trnh tinh ch v tinh sch protein. Sc k trao i ion : 75% Sc k i lc (AC): 60% Sc k lc gel (GF): 50% 2. Nguyn tc - Protein s gn thun nghch vi cc cht trao i bng tng tc ion gia cc nhm mang in tch tri du. - Sau , protein c chit ra khi tng dn lc ion lm tng tc ion b b gy. bi ny, t dng gradient mui NaCl t 1M Lu : C s ca IEC l s cnh tranh v tr ca cc nhm in tch vi cht cn tch gn vi cht trao i ion. N tu thuc vo in tch phn t v lc ion ca mi trng m. Khi nng ion cnh tranh cao th cc ion cn tch s ri khi cht trao i.

Hnh nh minh ho sc k tro i ion III. Thc hnh 1. Chun b dng c, ho cht - Ra sch, trng nc ct, sy kh cc dng c cn thit

- Pha dung dch m chy sc k Tris- HCl 50 mM, PH=8, c NaCl 50 mM (m A) 2. Chun b mu - Cn c 50g khoai ty, dng dao thi nh v nghin trong 50ml m A bng my xay sinh t. Sau trng my lc bng 50ml m A. Tng th tch m phi dng l 100ml. - Ly tm 10.000 rpm/20 pht thu dch trong - Nu dch vn c th tip tc lc thu dch trong bng giy lc. - Sau khi lc ta tin hnh o OD bc song A280 - a dung dch mu v pH =8 bng cch b sung tng lng nh m Tris ( kim tra pH bng giy qu) 3. Chun b gel - S dng gel DEAE-cellulose - Gel c chun b trc, loi b cc ht gel nh, v tri l lng. 4. Nhi ct gel - Ha gel trong 5 ln th tch m chy, lng, ht (gn) b cc ht gel nh l lng trn cho n khi cn 1,5 th tch. - Ra v trng sch ct, lp dy dn ra vo u di, gn ct ln gi theo phng thng ng. nh du v tr 5ml bng cch 5ml nc vo ri bm ra. - ng pha di ct bng cch dng bm. Khuy tht nh, ho u cc gel ri dng pipet 1000l (u cn ct to phn u) ht gel v th t t vo ct (trnh to bt kh)

- Khi ng bm cho dch m chy ra v gel lng xung di tc dng ca p sut trong ct. Tip tc thm gel vo thng xuyn cho n khi lng gel lng t th tch 5ml. - Khi gel n vch 5ml, cho chy qua ct t nht 5ml m vi tc khong 30-60ml/h. y l bc cn bng ct. 5. Gn mu ln ct - Sau khi c nng protein ban u tap pha long ra nng 20 mg/ml (cn ly 400 mg) n ta s ly 20ml dung dch mu. - Cho 20ml mu ny chy vo ct t t ngm xung ht - Ra protein khng gn trn ct bng m A. o OD280 < 0.1 th dng li. - Thu li 500l in di sn phm. 6. Thu mu - Ra chit mu bng gradient NaCl t 0.05M n 1M - S dng my thu phn on thu ln lt 70 ng nghim, mi ng 18 git mu. - Tin hnh o OD280 cc ng chn thu c theo th t 2,4,6,.70. ri thng k s liu theo bng. Kt qu - Protein = - Protein tng s = Dung dch chit th ban u em i pha long 10 ln ri o OD ta thu c ch s l 2,22(mg/ml) o Ta c: OD280 = 0.66 1mg protein/1ml dch mu. Protein tng s thu c l: = 660.5 (mg)

 Thu c bn s liu: IV. in di protein bng gel polyacrylamide c SDS 1. gel - gel polyacrylamide hai lp, gel c l 4% v gel tch l 12% - Phn gel tch c trc. Sau khi trng hp hon ton, phn gel c. - Gel c trng hp hon ton th sau 30-60 pht th s dng in di. 2. in di - Tin hnh in di: protein tinh sch, dch chit th, dch ln ct, FT, ra 10ml u, phn on 26, 40, 42, 60 - Trn 10l mu vi 30lm mu (nng 4x). t vo t m 90C trong 10 pht. in di mu 20l v marker 10l. - m chy in di l Tris-glycin, PH 8.8 cha SDS 1%. Chy in di vi in th n nh 150V.