3 Phng php in di trn gel polyacrylamide 2.3.

1 S lc v lch s gel polyacrylamide v s pht trin ca phng php in di trn gel polyacrylamide in di l mt phng php c s dng ch yu trong nghin cu sinh hc phn t. N cho php phn tch trng lng ca cc phn t sinh hc, t ta c th xc nh c trng lng phn t ca chng. Vo khong thp nin 1930 phng php in di trn gel u tin c bit n. Raymond v Winstraub (1959) ln u tin gii thiu v gel polyacrylamide. Ornstein v Davis (1964) thc hin in di trn gel polyacrylamide khng lin tc. Beber v Osborn (1969) gii thiu v tc nhn gy bin tnh protein v sodium dodecyl sulfate. Laemmli (1970) pht trin phng php SDS PAGE trong phn tch 28 thnh phn ca T4 phage. 2.3.2 Gel polyacrylamide Acrylamide l mt n phn t c cu trc: CH2=CHCONH2 v bisacrylamide c cu trc: CH2=CHCONHCH2NHCOCH=CH2. Acrylamide l mt c t thn kinh mnh, khi ht phi s lm m mt. N c kh nng thm qua da khi tip xc trc tip. Hiu qu c t s tch t dn. V vy khi thc hin lm th nghim vi acrylamide nn eo khu trang v gng tay. Tuy nhin dng c polymer ha thnh polyacrylamide th li khng c. Nhng khi tip xc trc tip vi n vn nn mang gng tay bo v v c th s cn mt lng nh acrylamide vn cha c polymer ha. (Sambrook v ctv, 2001) Gel polyacrylamide l gel c to thnh do s polymer ha cc n phn t acrylamide v N,N-methylene-bis-acrylamide. Kch thc l gel ph thuc vo chiu di ca chui polymer v mc polymer ha. Gel polyacrylamide c m t qua 2 c im: %C v %T. 1. Tng lng n phn t acrylamide cha trong gel (%T) %T= (g acrylamide + g bis- acrylamide) x 100% Tng th tch (ml) 2. Lng bis- acrylamide cha trong gel (%C) %C= g bis- acrylamide x 100% g acrylamide + g bis- acrylamide Qu trnh polymer ha c xc tc bi ammoniumpersulfate (APS), N,N,N,N-tetramethylethylenediamine (TEMED). S dng gel polyacrylamide chy in di th vic chun b gp nhiu kh khn hn so vi chy in di trn gel agarose. S di chuyn ca cc phn t sinh hc trn gel ph thuc vo kch thc ca l gel, in tch ca phn t cn phn tch v in trng. Gii hn trng lng phn t ca cc phn t sinh hc m gel c th phn tch ty thuc vo nng acrylamide v bis-acrylamide: nu nng gel thp s to ra l gel ln, cho php phn tch cc phn t sinh hc c kch thc ln v ngc li nu nng gel cao s to ra l gel nh, cho php phn tch cc phn t sinh hc c kch thc nh (xem ph lc 2). 2.3.3 Phng php SDS-PAGE (Laemmli, 1970) Phng php SDS- PAGE (Sodium Dodecyl Sulfate- Polyacrylamide Gel Electrophoresis) cn c gi l phng php in di trn gel acrylamide khng lin tc v mu protein c gy bin tnh (discontinuous and denaturing). SDS l mt tc nhn lm bit tnh v m tnh ha cc phn t protein. Trong k thut ny

protein c x l vi cht ty SDS v tc nhn kh cu ni disulfite l 2mercaptoethanol hoc dithiotheitol (DTT). Khi x l protein vi 2- mercaptoethanol hay dithiotheitol th cc tc nhn ny s ct t cc cu ni disulfite (S S) ca protein, v vy protein t cu trc bc 2, 3, 4 c bin i thnh chui polypeptide bc mt v nh s c mt ca SDS tt c cc protein u c tch in m. Nh , di tc ng ca in trng s di chuyn trong gel ca cc phn t protein ch ph thuc vo kch thc, nhng phn t c kch thc ln s di chuyn chm hn nhng phn t c kch thc nh khi i qua mt l gel c kch thc nht nh. Di tc dng ca in trng cc phn t tch in m s di chuyn v cc dng. a cc protein trong mu v cng mt vch xut pht ng thi to ra s phn tch tt hn, ngi ta thng s dng phng php in di trn gel khng lin tc (discontinuous gel). Trong phng php ny gel gm 2 lp: Lp gel gom (stacking gel): Thng thng lp gel ny c nng gel thp vo khong 4 5%, v nm pha trn ni to ging bm mu. Khi c tc ng ca in trng cc protein c trong mu s bt u di chuyn t nhng ging mu qua lp gel ny. Nh , cc protein trong mu c dn li v to mt lp bng mng nm ngay pha trn lp gel phn tch, to iu kin thun li hn trong vic phn tch protein [5]. Lp gel phn tch (separating gel) (lp di): Lp gel ny c nng gel cao hn so vi lp gel gom, v nm pha di. C tc dng chnh trong vic to ra cc bng protein c trng lng phn t, kch thc khc nhau t mt hn hp protein xut pht ban u [5]. K thut SDS- PAGE c th xc nh c nhng phn t protein c trng lng phn t t 10.000 200.000 Daltons. Nhng phn t protein c trng lng ln hn 200.000 Daltons thng c xc nh trn gel c nng acrylamide t hn 2,5% [5]. 2.3.4 Nhum gel sau khi in di Nu protein khng c nh du phng x, th sau khi in di thy c s phn tch ca cc bng protein trn gel chng ta cn phi tin hnh nhum gel. S la chn phng php nhum c xc nh da trn nhiu yu t bao gm nhy, gii nhum, tin dng, tnh kinh t, thit b nhum, thit b c sn c. Coomassie Brilliant Blue R- 250 thch hp dng nhum cho hu ht cc protein v thng c s dng. i vi Coomassie Brilliant Blue G- 250 nn s dng nhum i vi nhng protein c trng lng phn t polypeptide thp. Nhum bc ... l phng php rt nhy i vi protein v nn s dng nhum gel khi in di nh gi s tinh khit ca mu protein. Nhum bc cho php pht hin mt cch nhanh chng v nhy cc bng protein trn gel ngoi tr nhng protein khng c phn tch trn gel. a) Nhum Coomassie Brilliant Blue Nhum bng Coomassie Brilliant Blue R- 250 l phng php nhum ph bin nht thng c s dng pht hin protein trn gel polyacrylamide (Wilson, 1983). Rt d pht hin khi c khong 0,1 1 g protein trn bng. Dung dch nhum bao gm cc thnh phn nh: 0,1% Coomassie Brilliant Blue R- 250 (w/v), 40% methanol (v/v), 10% acid acetic (v/v). Ty theo hm lng protein m ta c th thay i t l thnh phn cc cht cng nh cht nhum. Nu trn gel cha polypeptide c trng lng phn t thp th ta c th nhum trong hn hp dung dch 0,1% Coomassie Brilliant Blue R- 250 (w/v), 50%

methanol (v/v), 10% acid acetic (v/v) khong mt gi. Ngoi ra, chng cng c th c nhum vi 0,025% Coomassie Brilliant Blue G- 250 (w/v) trong methanol v acid acetic t 1 2 gi. b) Nhum bc Phng php ny c th pht hin protein nhy gp trm ln so vi phng php nhum bng Coomassie Brilliant Blue (Merril, 1990). Nhng bng cha khong 10 100 ng protein hay nucleic acid c th d dng c nhn thy. Thng thng ngi ta s dng mui bc nitrate. Cc bc tin hnh nhum bc phc tp hn rt nhiu so vi nhum bng Coomassie Brilliant Blue [9]. Cc bc nhum (Kyle Coachman, Jeff Ranish v Steve Hahn, 2002) 1. Ngm gel trong acid acetic 7% khong 10 pht. 2. Ngm gel trong methanol 50% khong 20 pht. 3. Ra gel trong nc siu sch khong 10 pht. 4. Chun b dung dch nhum: Chun b dung dch A gm: 0,8 g bc nitrate, 4 ml nc siu sch.Chun b dung dch B gm: 21 ml nc siu sch, 250 l NaOH 30%, 1,4 ml NH4OH 14,8 M. 5. Nh t t tng git dung dch A vo dung dch B. Ngay lp tc thm vo hn hp dung dch 76 ml nc siu sch. 6. Ngm gel trong dch nhum khong 15 pht. 7. Ra gel tht k li vi nc siu sch. 8. Ngm gel trong dch gii nhum (200 ml nc siu sch, 1 ml citric acid 1%, 100 l formaldehyde 37%) cho ti khi c th thy r cc bng protein. Thng thng t 2 15 pht. 2.3.5 Mt s yu t cn quan tm trong in di trn gel polyacrylamide a) H m in di v cc tc nhn gy bin tnh protein H m quyt nh nhu cu v nng lng v nh hng n s phn tch protein. H m bao gm m dng pha gel v m dng chy in di. H m ca gel khng lin tc u tin c pht trin bi Ornstein (1964) v Davis (1964). H ng dng phn tch protein nguyn th, tc l nhng protein cha b bin tnh. H m h s dng gm bn yu t: (i) gel gom dng m Tris HCl pH 6,8; (ii) gel phn tch dng m Tris HCl pH 8,8, (iii) m in di Tris glycine pH 8,3; (iv) mu cn phn tch dng m Tris HCl pH 6,8. Nhng vi h m c s dng nh vy th trong m hnh ny h khng th tin hnh phn tch c mt khong rng v trng lng protein. Da trn m hnh v h m ca Ornstein v Davis, Laemmli pht trin trong vic s dng thm tc nhn kh cu ni disulfite v SDS. Nh , vic phn tch protein trn gel ch ph thuc vo trng lng phn t ca protein. Do vic phn tch protein tr nn d thc hin hn v khng b ph thuc vo in tch ca cc phn t sinh hc. Tuy nhin m hnh ca ng cng gp mt s kh khn. Nhiu protein khng c phn tch theo mong mun khi s dng SDS PAGE (Andrews, 1986; Hames, 1990; See v Jackowski, 1989). Hin tng ny xy ra l do: (i) cu trc disulfite ca protein khng b tc ng, v protein khng b m tnh ha bo ha bi SDS. (ii) Nhng glycoprotein v lipoprotein trong mu khng b m tnh ha bo ha bi SDS, bi v thnh phn khng phi protein ca chng khng tng tc vi SDS. b) iu kin nng lng

Nng lng cung cp trong in di l in nng. C rt nhiu thit b ngun in khc nhau c sn xut phc v trong in di. Mt s thit b cho php chy t ng sau khi ta chnh cc thng s mong mun (hiu in th, cng dng in, thi gian in di) v cng c mt s thit b i hi phi c s gim st v thi gian (Allen v ctv, 1984). Gii hn chnh ca cc m hnh in di l kh nng lm tht thot nng lng khi in nng chuyn ha thnh nng lng in trng. S tht thot l do mt phn nng lng in c chuyn ha thnh nhit nng (Wooolley, 1987). Nhit nng ny c th gy ra mt s nh hng xu n kt qu in di nh: bng protein b mo, cong; tng s khuch tn; s hot ng tr li ca enzyme trong mu; s bin tnh protein. Do mt thit b in di tt phi m bo c s chuyn nhit t gel ra mi trng ngoi. Thng thng, in di nn chnh hiu in th (volt) v cng dng in (ampe) sao cho qu trnh in di xy ra nhanh chng m vn m bo c s phn tch protein mu. 2.3.6 Phng php in di hai chiu K thut in di hai chiu trn gel polyacrylamide (2 DE) l mt k thut c kh nng phn tch hn 1.800 protein trn cng mt bn gel, n l mt cng c quan trng c bn trong nhng th nghim m cc protein phc tp c tch ra phn tch ng thi.Cc bc c bn trong k thut 2 DE: Chun b mu v lm ha tan mu: Phng php chun b mu ty thuc vo mc ch nghin cu. Cc yu t nh tan, in tch, kch thc v im ng in ca protein u c lu trong qu trnh chun b mu. Vic chun b mu cng gip lm gim bt s phc tp ca mt hn hp protein. Cc on protein c dng trong chy in di hai chiu phi c duy tr trong dung dch m bin tnh c lc ion thp duy tr tnh nguyn vn ca protein v gi chng trng thi ha tan. in di theo chiu th nht: Protein c tch ra da trn im ng in pI ca chng, pH m protein khng tch in v khng di chuyn trong in trng. K thut ny c gi l isoelectric focusing (IEF). i vi in di hai chiu, IEF l cng c tt nht thc hin phn tch protein theo nng pH. Protein l mt phn t ion lng tnh. Khi m protein c t trong mi trng vi gradient pH v b ph thuc vo mt trng in t, ban u n s di chuyn n in cc tri du vi n. Trong sut qu trnh tn ti ca chng qua gradient pH, protein s c th nhn hay mt proton. Cn bng trng thi ca protein: trng thi bnh thng protein c th mang in tch m, in tch dng hay cn bng v in tch. iu ph thuc vo s lng nhm ( NH2) v (COO ) ca cc phn t amino acid cu to nn protein. Do , trc khi thc hin in di theo chiu th hai, cc phn t protein cn phi c gy bin tnh v ng nht v in tch. bc ny ngi ta x l protein vi 2-mercaptoethanol hay dithiotheitol, cc tc nhn ny s ct t cc cu ni disulfite (SS) ca protein, v vy protein t cu trc bc 2, 3, 4 tr thnh chui polypeptide v nh s c mt ca SDS tt c cc protein u c tch in m. Phn tch protein theo chiu th hai: Di tc dng ca in trng do protein mang in tch m sau khi cn bng trng thi nn protein s di chuyn t cc m sang cc dng. Khi protein di chuyn qua gel polyacrylamide, s c st ca cc ht acrylamide vi phn t protein to ra lc khng lm ngn cn s di chuyn ca protein. Protein c trng lng phn t cng ln th lc cn cng mnh.

Nhum protein: c th nhn thy c protein sau khi in di, gel phi c nhum, tr khi chng ta nh du protein. S la chn phng php nhum c xc nh da trn nhiu yu t bao gm nhy, di nhum, tin dng, tnh kinh t, v thit b nhum, thit b c sn c. Thu nhn v phn tch hnh nh protein: Vic thu nhn hnh nh theo k thut s l mt trong nhng yu t quan trng lm cho phng php 2-DE l phng tin thc t trong vic thu nhn d liu trong nghin cu. Phn mm phn tch protein PDQuest (Bio Rad) cho php chng ta phn tch c cc hnh nh gel, ch gii cc im protein v thu nhn d liu. Phn mm PDQuest cng tham gia iu khin cc thit b ghi nhn hnh nh v h thng thu thp protein im ca h thng thit b phn tch protein Spot Cutter. 2.4 Mt s nghin cu ng dng in di protein SDS- PAGE 2.4.1 Nghin cu trong nc ng dng in di protein SDS- PAGE Nm 2005, Trn Linh Thc (i hc Khoa hc t nhin Thnh ph H Ch Minh) s dng phng php SDS- PAGE trong ti Nghin cu cc h thng biu hin gen trong E.Coli sn xut protein ti t hp chng minh s biu hin ca gen v s hin din ca protein ti t hp. Nm 2005, nhm nghin cu (Khoa Nng nghip v Sinh hc ng dng, trng i hc Cn Th nghin cu ng dng phng php in di protein SDSPAGE nh gi thun ca mt s b ht ging rau cc loi. Ht ging rau a vo nh gi l loi ht lai F1 c sn xut trong nc v nhp khu. Nm 2005, V Th Hong Mi (i hc M Bn cng Thnh ph H Ch Minh) s dng phng php SDS- PAGE kt hp vi phng php Western blot trong ti Cc phng php chun on thng hn thng dng vi mc ch phn tch cc thnh phn protein khng nguyn S. typhi. Nm 2007, V Cng Thnh v ctv (Khoa Nng nghip v Sinh hc ng dng trng i hc Cn Th) ng dng k thut in di protein SDS-PAGE ci thin thnh cng phm cht nhiu ging la c sn ang b thoi ha ca ng bng sng Cu Long. Trong chn to ging la, k thut ny gip pht hin nhanh nhng tnh cht ni bt nh mi thm, protein, amylose cc nh khoa hc chn lc c nhng dng, ging c phm cht tt. Mt s ging la c sn c ci thin phm cht thnh cng v nhiu ging la trin vng ra i bng k thut ny. Nm 2007, Phm Vn Phng (i hc Cn Th) ng dng k thut in di SDS-PAGE protein nghin cu c im di truyn v chn ging la. K thut in di SDS-PAGE protein gip nh chn ging sm tuyn chn chnh xc nhng c th mong mun ngay t th h u ca cc t hp lai nn rt ngn thi gian tuyn chn so vi phng php truyn thng. K thut in di SDS-PAGE protein gip nh gi a dng di truyn v phn bit hai loi la hoang ng bng sng Cu Long da vo mc n mu Coomassie ca bng protein ch th dng basic glutelin. 2.4.2 Nghin cu ngoi nc ng dng in di protein Fan Shuguo (1999) ng dng in di protein nh gi tnh chu mn khc nhau ca mt s loi cy trng. Nazrul Islam, M. Lonsdale, N. M. Upadhyaya, T. J. Higgins, H. Hirano v R. Akhurst (2004) thc hin nghin cu ti ly trch protein t l la cho in di hai chiu v ng dng n trong phn tch h protein ca la. Scott A. Young v ctv (1995) thc hin nghin cu s tch ly peroxidase

trong mch g ca la trong sut qu trnh phn ng khng tng thch vi Xanthomonas oryzae pv oryzae. H bt u chng X. oryzae pv oryzae cho la khi la c 2 3 l. Sau ct ly nhng mu l khi chng c 48 gi, tin hnh ly trch protein ca l sau tin hnh in di trn gel polyacrylamide trong iu kin protein mu khng b bin tnh (Discontinuous and nondenaturing). Chn ly bng protein c kch thc tng ng vi peroxidase (42 kD), tip tc tin hnh in di SDS- PAGE v lai trn mng PVDF. Vch protein s c pht hin trn mng nh nhum CBB. Sau ra v thu mu gii trnh t. - See more at: http://congnghesinhhoc24h.com/tai-lieu/thiet-lap-quy-trinh-dien-di-protein-sds-page-va-ungdung-danh-gia-phan-427.html#sthash.UPflUV5q.dpuf