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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

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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

4. MI TRNG CHT MANG S DNG TRONG IN DI

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Cht mang

u im

Nhc im

Mi trng cht mang

Nguyn liu dng lm cht mang trong in di: giy lc, acetate cellulose, agarose, polyacrylamide, lp mng silica, alumina, Phn loi da vo s khc nhau ca mi trng in di: in di trn giy (paper electrophoresis) in di trn bn gel (gel electrophoresis) in di trn ng vi qun (capillary tube electrophoresis)

Giy

Cha t nhm c mang in tch Lun c sn D cm, thao tc (khng cn x l)

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

PolyThng c s dng acrylamide p dng cho protein v acid nucleic

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

4.1. in di trn giy

Phn tch cc phn t c MW nh
VD: amino acid, protein c kch thc nh

4.1. in di trn giy

Giy lc ct thnh nhng dy bng c chiu rng 3 5 cm

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4.1. in di trn giy (tt)

u im: r, kinh t, tin dng Nhc im: Kt qu khng chnh xc Tn thi gian Kh p dng cho mt s loi phn t nh protein hoc nhng i phn t c tnh thn du (hydrophilic) v giy s hp thu cc dy band b ko thnh vt di

4.2. Acetate cellulose

C tnh hp thu t hn giy

4.2. Acetate cellulose

Khi p dng cho cc loi phn t nh protein hoc nhng i phn t c tnh thn du (hydrophilic) cho kt qu l cc dy band sc nt, khng c hin tng b ko di thnh vt t tin nhng vn c s dng rng ri trong cc PTN y khoa trong vic phn tch nhng mu protein (nh lng protein huyt thanh)

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4.3. Mao qun (capillary)

Vi qun: silica nng chy, c tnh mm do nh c bc polyimide.

4.3. Mao qun (capillary) (tt)

Din tch mt ct ngang nh tiu th nng lng ti thiu, kh nng tch cht nhanh (ch trong vi pht) H thng t ng ha, ghi nhn kt qu bng my ghi (sc k ) khng phi nhum mu bn gel

4.3. Mao qun (capillary) (tt)

t tin khng loi b ct, phi ra k, loi b hon ton cc mu ln trc, ti s dng 5 50nl ( 1% in di c in) Cn u d cc nhy pht hin tn hiu

4.3. Mao qun (capillary) (tt)

Phn tch h gen, sng lc cc bnh Pht hin tc nhn gy bnh, nh chng Pht hin cy trng chuyn gen (GMO), cc sn phm trong nng nghip ....

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4.4. Gel agarose

Polymer (polysaccharide) trch t loi rong Khng mang in tch C kch thc l ln hn so vi gel acrylamide u im: khng cn thm cht ph gia v cht to mng ngang polymer ha, khng c hi, r tin Dng tch cc phn t ln (DNA)

4.5. Gel polyacrylamide

c

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

4.5. Gel polyacrylamide (tt)

C th iu ch gel mt nng nht nh hoc c nng tng dn tuyn tnh (xc nh trng lng phn t, im ng in) Nng ca acrylamide s to gel c l rng kch c khc nhau <2.5%: tch protein c trng lng phn t ~ 1.000 KDa >30%: tch protein c trng lng phn t ~ 2 KDa

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5. MT S K THUT IN DI TRN GEL POLYACRYLAMIDE

in di trong iu kin bin tnh (SDS-PAGE) in di trong iu kin t nhin

5.1. in Di Trong iu Kin B Bin Tnh

Phn tch cc protein km ha tan in di vi gel polyacrylamide c cha SDS (sodium dodecyl sulphate polyacrylamide gel electrophoresis, SDS PAGE)

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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

tch cc i phn t da vo kch thc phn t

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Mi lin quan gia T% ca gel v trng lng phn t ca hp cht in di

T% Trng lng phn t ca hp cht in di <2.5 1.000.000 5 25.000 300.000 7 100.000 10 15.000 70.000 15 12.000 50.000 30 2.000

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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

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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

5.2. in di trong iu kin t nhin

in di vi h thng bt lin tc p dng vi cc mu protein ha tan v khng b kt ta trong qu trnh in di Mi trng in di c pH bt lin tc, gm 2 lp gel khc nhau c pH ban u hon ton khc nhau, c t lin k nhau

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

6. in di hi t (Isoelectric focusing, IEF)

im ng in (isoelectric point, pI): gi tr pH m protein khng cn in tch

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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

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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 o im ng in (IMF): gel c dng hnh ci que

Mu phn tch c t ln 1 u que, mi que cha 1 mu, in di nhiu que 1 lc

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

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7.2. CC THIT B S DNG TRONG SDS PAGE 2 CHIU

Thc hin IEF trn tm gel t trong hnh ng tr lm bng thy tinh, ng knh ng 5mm, chiu di 14cm Sau khi thc hin IEF xong, que gel c tht tut ra khi ng cha, ri c t trn mt agarose m v khi ngui li, que gel gn dnh vo tm agarose. Tm agarose ny c t nm ngang, phn lp gel chun b ca mt tm gel SDS-PAGE

8. Hin hnh bn gel

sau khi in di, bn gel cn c lm c nh, ngha l lm cho cc protein kt ta, c nh trn bn gel v ng thi loi b nhng thnh phn khng phi l protein v cc thnh phn ny c th gy tr ngi cho qu trnh hin hnh bn gel Phng php c nh tt nht: ngm bn gel trong dung dch 10 20% w/v acid trichloroacetic hoc hn hp acid trichloroacetic v acid sulphosalicylic (10% w/v cho mi cht), ngm nhit phng trong khong 2 gi

8. Hin hnh bn gel (tt)

Thuc nhum Amino black Coomassie blue Ponceau red (thuc nhum thun nghch) Bis-1-anilino-8-napthalen sulfonate (ANS) Nile red SYPRO orange Flourescamin AgNO3 SYPRO red Loi cht pht hin Protein Protein Protein Protein Protein Protein Protein Protein/DNA Protein Gii hn pht hin 400 ng 200 ng 200 ng 150 ng 20 ng 10 ng 1 ng 1 ng 0.5 ng

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8.1. Nhum mu bn gel bng Coomassie Brilliant Blue

Protein hin hnh mu xanh dng u im:
Phm mu khng xuyn qua h thng mng gel nn qu trnh ty bn gel xy ra nhanh chng Thao tc n gin

8.2. Nhum mu bng dung dch bc nitrat

Ngm bn gel trong dung dch AgNO3 cho kt ta kim loi bc ti cc vng c s hin din ca protein hoc DNA to thnh vng mu en u im: nhy cao gp 20 200 ln so vi CBB (0.05 0.1ng protein/mm2 gel, c th pht hin cc protein lng vt Nhc im Cc protein nh Ig dy ngn cho hin mu rt km t tin, tn nhiu thi gian

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

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9. Western blotting (tt)

Lai protein (antigen) vi khng th (antibody - Ab) c hiu Nghin cu protein v enzyme

9.1. Khng nguyn v khng th

Khng nguyn: cc cht min dch Hp cht c trng lng phn t nh (cc phn t ng) Hp cht polymer sinh hc c cu trc phc tp (protein) Khng th: protein nhn bit khng nguyn

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

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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,

Bi ging SHPT & KT Gene _ Hutech

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Bi ging SHPT & KT Gene _ Hutech

Bi ging SHPT & KT Gene _ Hutech

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