Professional Documents
Culture Documents
L/O/G/O
1. Khi nim
K thut tch nhng hp cht ca mt hn hp nh in trng VD: tch hn hp cc amino acid, nucleic acid, protein
IN DI
2. Nguyn tc
Cc protein c mang in tch khi n mi trng c pH khc vi pI Cc ion mang in tch ngc du nhau s di chuyn theo 2 hng ngc chiu nhau Cc ion mang in tch cng du di chuyn vi vn tc khc nhau Cc phn t tch ri nhau
3. Cn bn vt l ca in di
Vn tc di chuyn ca 1 phn t c in tch c m t theo phng trnh
v=
E!q f
Trong : v: vn tc di chuyn ca phn t trong in trng E: lc in trng q: in tch ton phn ca phn t f: h s ma st
2/13/12
3. Cn bn vt l ca in di (tt)
in trng c thit lp bng cch p mt in th V vo 2 in cc, t cch nhau 1 khong cch d Lc in trng s l E = V/d
3. Cn bn vt l ca in di (tt)
in tch ton phn:
Tng s in tch m v in tch dng trong phn t
H s ma st (f) ph thuc:
Khi lng phn t Mc cht ch trong cu trc phn t nht ca dung dch m rng ca h mng mi trng in di
3. Cn bn vt l ca in di (tt)
Phn t cng c nhiu in tch v kch thc cng nh th di chuyn cng nhanh v in cc ngc du. Cc phn t c cng in tch nhng hnh dng khc nhau s di chuyn vi vn tc khc nhau
2/13/12
Cht mang
u im
Nhc im
Giy
nhy thp Tnh lp li thp Khng kim sot c kch thc cc l rng c hi cho thn kinh Gel c khong i sng ngn Khng gp xp bn gel c
Agarose
Khng cn thm ph gia Khng t tin Dng cho DNA, RNA Cn u d nhy cm Dng c t tin Mun ti s dng phi mt thi gian
Mao qun C th ti s dng. Kh nng tch cht cao. Phn tch nhanh Tn lng mu t D p dng h thng t ng
2/13/12
2/13/12
2/13/12
S2O82- 2SO42Bn c hc, tr ha hc C th thay i rng tch nhng phn t c cng in tch nhng kch thc v hnh dng khc nhau
2/13/12
2/13/12
5.1. (tt)
SDS hp cht c dy alkyl di 12C, c tnh k nc v 1 u mang nhm sulfat phn cc cht hot ng b mt c kh nng ha tan rt hiu qu hu ht cc loi protein
Protein c SDS bao bc u bn ngoi, thnh 1 lp che ph ng nht, tch in m SDS loi b s khc bit v in tch gia cc i phn t trong dung dch Cc phc protein-SDS c in tch ton phn ging nhau
2/13/12
2/13/12
5.1. (tt)
u im K thut n gin Kt qu c tnh lp li cao Kh nng tch cht rt tt: tch cc protein c dch chuyn chnh lch nhau 1% = phn bit c s khc nhau 1KDa gia nhng protein c trng lng phn t khong 100KDa
10
2/13/12
5.1. (tt)
Nhc im Kh tch cc polypeptide <10KDa Kh phn bit cc protein c cng kch thc hoc kch thc gn ging nhau Protein khng gi c hot tnh sinh hc
5.1. (tt)
in di vi h thng bt lin tc (tt) Stacking gel (gel chng ng mu, gel chun b) lp gel pha trn, c chiu cao ~ 1/5 tng chiu cao bn gel Nng polyacrylamide long (3 5%), pH trung tnh (6.9) Gel c l rng to, lc ion yu Resolving gel (gel phn tch mu) Lp gel pha di, chiu cao 4/5 bn gel Nng polyacrylamide c (8 20%), pH base ( 8 9) Gel c l rng nh, lc ion mnh
11
2/13/12
6. (tt)
Gi tr im ng in ca mt s protein
Protein Pepsin Pepsinogen ovalbumin Bovine serum albumin urease pI 1.0 2.8 4.6 4.9 5.0
6. (tt)
Mun xc nh pI, cn phi thit lp 1 gel vi gradient pH n nh
S dng cht in ly lng tnh: hp cht tng hp, sn phm ca s polymer ha cc loi acid oligoamino (-NH2) oligocarboxylic (COOH) c MW nh Trn cc hp cht in ly khc nhau (khng c s hin din ca in trng): hn hp ch c 1 pH duy nht Khi c dng in, tng cht ring l di chuyn n v tr pI ca n chng hot ng nh dung dch m v c pH gradient
6. (tt)
in di hi t c pH tuyn tnh nh cc cht bt ng
S dng nhng hp cht l dn xut ca acrylamide (CH2=CH-CONH2, cht bt ng), trong cu trc c cha cc nhm acid yu (CH2=CH-CONH-COOH, acid carboxylic), hoc base yu (CH2=CHCONH-NH2, amin tam cp)
6. (tt)
u im: Loi gel ny tng thch c vi urea v cc cht to nh Khng b nh hng bi cc mui cha trong mu protein
12
2/13/12
ng dng ca IEF o pI
Khi mt protein c t vo bn gel gradient pH, protein ny di chuyn v v tr pI ca n Nu protein trong mi trng c pH cao hn gi tr pI ca n, protein c in tch ton phn l in tch m, v di chuyn v pha anod v ngc li Ch mi trng pH = pI, protein c in tch ton phn = 0 (zero net charge) v khng di chuyn trong in trng
7. in di SDS-PAGE 2 D
7.1. Nguyn tc Protein c tch theo chiu th nht bng in di o im ng in (da vo s khc bit v gi tr im ng in) v chiu th 2 bng SDS-PAGE (da vo s khc bit v MW)
in di SDS-PAGE:
Cc que gel c tht tut ra khi ng cha, ri t ln trn 1 tm agarose m Khi agarose ngui, que gel gn dnh vo tm agarose Tm agarose c t nm ngang, pha trn u gel SDS-PAGE in di SDS-PAGE theo chiu c gc 90o so vi chiu thc hin IEF
13
2/13/12
14
2/13/12
Nhc im
Phm mu km nhy, ch pht hin khong 0.5g protein/cm2 gel
9. Western blotting
K thut in thm (electroblotting): p dng in trng vung gc vi bn gel Protein >15kDa: mng nitrocellulose vi ng knh l rng 0.45 m Protein <15kDa: mng nitrocellulose vi ng knh l rng 0.2 m
15
2/13/12
9.1 (tt)
1 protein v 1 khng th gp nhau Tng tc c trng xy ra gia vng bin i (variable region) ca khng th v epitope (vng c bit trn khng nguyn) ca khng nguyn Cc epitope khc nhau trong nhng cu trc protein c th nhn din bi cc Ab c trng PAb: nhn din nhiu epitope trong cng 1 Ag MAb: nhn ra 1 epitope
9.1 (tt)
Vi khng th a dng (polyclonal antibody): mt s epitope hin din trn b mt ca 1 antigen Khng th n dng (monoclonal antibody, Mab): 1 epitope hin din trn 1 antigen
16
2/13/12
9.1 (tt)
S dng lng tha Ag hoc Ab, sau khi nhum mu phc to thnh gia Ag-Ab o nng ca Ag hoc Ab
9.2. Nguyn tc
Tch chit protein in di Chuyn ln mng lai Ra mng lai vi dung dch kha (dung dch protein, blocking solution): kha v tr khng c trng trn khng th Lai protein trn mng vi cc protein khng th c hiu (VD: IgG th: gn vo epitope ca protein)
9.2. (tt)
Khng th ny s c pht hin nh khng th th 2, c i lc c trng vi khng th th nht (VD: IgG khng th) Lm hin mu protein: Hin hnh pht hunh quang: cho tc dng vi fluorescein isothicyanat Ghi du phng x Phn ng enzyme: s dng perixidase, galactosidase,
17
2/13/12
18