SC K GEL - NG DNG

TRNG I HC BCH KHOA TP.HCM KHOA K THUT HA HC B MN HA HA HU C

A

BK

TIU LUN CC PHNG PHP PHN TCH CNG C

TN TI:

Thnh ph H Ch Minh, ngy 7 thng 1 nm 2014

SC K GEL - NG DNG

MC LC

GII THIU3L THUYT CA QU TRNH SC K GEL5c im ca qu trnh5ng cong chn lc v la chn phngphp8 gii li9PHN LOI GEL11Superdex11Sephacryl13Superose15Sephadex16Sephadex LH-2018NG DNG19

IV.1.Kh mui - i dung mi - lm tng nng protein19

IV.2.Sc k gel trong chin lc tinh ch21

IV.3.Xc nh khi lng phn t v phn tchs phn b khi lng phn t .

26

TI LIU THAM KHO30SC K GEL - NG DNG

SC K GEL - NG DNG

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GII THIU

Sc k gel dng tch cc phn t da trn s khc nhau v kch thc ca cc phn t. Nhng phn t khng b gi li bng lin kt ha hc nn thnh phn ca dung mi gii li (buffer) khng nh hng trc tip n gii li (resolution). Sc k gel ph hp vi nhng phn t sinh hc nhy cm vi pH, nng ion kim loi, mi trng khc nghit.

Qu trnh phn tch bng phng php sc k gel (gel filtration)

Gel c nhi vo ct to thnh m (packed bed).

m l mng li l xp hnh thnh t ht hnh cu c tnh bn vt l, bn ha hc v tr.

Lp gel cn bng vi dung dch i qua (buffer). Dung dch in y vo mng li l xp v khong khng gia cc ht.

Cht lng bn trong l xp c xem nh l pha tnh v cht lng bn ngoi c xem l pha ng.

ht gel hnh cu

2. mu c a vo ct

Pha ng ko mu di chuyn qua ct. Phn t s khuch tn vo v ra khi l xp, nhng phn t nh i vo su trong l xp v b gi li trong ct lu hn

Hnh I.1. Qu trnh sc k gel

Pha ng qua ct lin tc, cc phn t ln khng th chui vo cc l xp nn i ra khi ct trc. Nhng phn t nh khuch tn vo v ra khi l xp nn ra khi ct sau.ng dng ca phng php sc k gel Tch loi b nhng nhm phn t (Group separation)

Loi b nhng phn t nh nh mui, protein nh du ra khi nhm cc phn t ln. Thng c s dng tinh ch protein kt hp kh mui v i dung mi. Sephadex G-10, G-25, G-50 c s dng cho nhm ny Phn tch vi gii li cao (High resolution fractionation)

Phn tch nhiu cu t trong mt mu da trn s khc nhau v kch thc. Mc ch l phn lp mt hoc nhiu cu t xc nh khi lng phn t hoc phn tch s phn b khi lng ca mu.

Ph hp cho bc tch cui cui cng ca vic tinh ch. Monomer c th c tch khi ta v mu c th thay i dung mi ph hp cho th nghim hay d tr.L THUYT CA QU TRNH SC K GELc im ca qu trnh

Kt qu ca qu trnh sc k gel thng c biu din trn gin gii li hay ph sc k cho thy s khc nhau v nng (lin quan n mc hp thu tia UV vng 280 nm) ca cc thnh phn trong mu khi chng c gii li khi ct da trn trt t v kch thc phn t. Trn gin chia ra lm 3 loi phn t. Nhng phn t khng vo mng li m m c gii li thng ra khi ct vi cng tc ca dng lu cht ca pha ng (buffer) th tch Vo (xt trn 1 n v th tch ct), gi l th tch khong trng (void volume). Nhng phn t c mt phn i vo mng li m silicagel, ri mi gii li ra khi ct theo trt t kch thc nh hn s ra trc. V cn li l nhng phn t c gi hon ton trong mng li l xp v i ra khi ct trc gi tr tng th tch ct Vt (total column volume) ca pha ng i qua ct.

high

Absorbancemolecular

weight

Column Volumes (cv)

Hnh II.1. Ph sc k

Trng thi ca mi cu t c biu din lin quan n th tch gii li ca chng, Ve (elution volume) c o trc tip t ph sc k. C 3 cch xc nh Ve:

*290nm

A,_

Khi th tch ca mu qu nh

so vi th tch lp m th Ve

c xc nh t v tr ban u

ca mu n v tr cao nht ca

mi sc k.

ve

B

Khi th tch ca mu ln, khng th b qua so vi th tch lp m th Ve c xc nh t v tr mt na th tch mu n v tr cao nht ca mi sc k.

c

Khi th tch mu qu ln, to nn mi sc k c vng phng ngang, th tch Ve c xc nh t v tr bt u ca mu n v tr ca im un phn ang tng ln ca mi gii li.

Elution Ylume Hnh II.2. Cch xc nh th tch gii li Ve

Bi v mi i xng ph bin trong sc k gel, do Ve d dng xc nh. Tuy nhin Ve khng hon ton phn nh cch thc di chuyn ca cc cu t khi qua ct bi v Ve s thay i ph thuc vo Vt tng th tch ca lp m v cch nhi ct. S gii li ca mu c c trng bi h s phn b Kd.

gel matrix

Kd c trng cho phn pha tnh m xy ra s khuch tn ca nhng loi phn t khc nhau.

Cch xc nh Ve c nu trn.

Vo l th tch khong trng, chnh l th tch ca nhng phn t trong pha ng khng chui vo c mng li l xp do kch thc ln v di chuyn thng qua ct bng th tch ca pha ng (buffer). Ct c nhi tt th th tch Vo xp x 30% th tch ct.

Vs l th tch pha tnh bng Vi l th tch pha ng bn trong mng li (ch cha cc phn t kch thc nh). Trong thc t Vi rt kh xc nh do phi xc nh th tch mng li gel (th tch vt liu rn hnh thnh mng li), do thun tin th V; c thay th bng Vt - Vo.

Hnh II.3. Biu th hin th tch Vo v Vt

Do thay V; bng Vt - Vo, do ta c gi tr Kav, n khng thc s ng vi gi tr h s phn b Kd. Nhng i vi mi loi gel ring th c t l gia Kav:Kd, v t l ny khng ph thuc bn cht v nng ca mu.

Ve-K

Knii

Vt - vn

Hnh II.4. Mi quan h gia cc biu thc c s dng qu trnh gii li

ng cong chn lc v la chn phng php

H s phn b Kav lin quan n kch thc ca phn t. T trng v kch thc ging nhau ca cc phn t m t mi quan h gia gi tr Kav v log Mr (khi lng phn t). Trn mt vng xem xt th mi quan h ny l ng thng. Chn la phng php sc k gel ch ph thuc hon ton vo s phn b kch thc l xp v c m t bi ng cong chn lc. Bng cch v ng cong i qua nhng im gi tr Kav ng vi log Mr ca cc protein chun c th xy dng c ng cong chn lc.

Phng php lc gel c chn la sao cho khi lng phn t ln c gii li th tch khong trng Vo (Kav = 0) vi mi gin rng t nht v thi gian l ti thiu. Nhng hp cht c khi lng phn t nh nht c gii li gn gi tr Vt (Kav = 1)

Nu Kav > 1, c th phn t b mc kt trong mng li gel do kch thc qu nh (Ve > V)Nu Kav < 0, c th trong ct xut hin nhng knh, rnh m cc phn t c th chy tt qua ct (Ve < Vo).

Hnh II.5. ng cong chn lc ca Superdex 30 prep grade, Superdex 75 prep grade v

Superdex 200 prep grade

gii li

gii li cho bit phn tch gia cc mi, chu nh hng bi nhiu yu t nh t l gia th tch mu v th tch ct, tc dng, thng s ct, kch thc ht, s phn b kch thc ht, t trng lp m, xp ca ht, nht ca pha ng. Hiu qu ca qu trnh sc k gel ph thuc vo ch yu vo cc iu kin la chn sao cho chn lc hp l v trnh nhng mi gin rng trong qu trnh phn tch.

gii li c xc nh bng biu thc:

^r2 1

Rs =

W^ + w2^

Vr1 v Vr2 l th tch gii li ca 2 mi gn nhau.W1 v W2 l rng tng i ca mi.

Hnh II.6. Thng s c s dng xc nh gii li

CDCD

gii li l hm ca chn lc ca phng php v hiu qu ca phng php to ra nhng mi hp.

Hnh II.7. S ph thuc ca gii li vo chon lc v rng ca mi

1oo

Hiu qu ca ct nhi ph thuc vo vic to ra mi hp i xng trong qu trnh gii li v c xc nh da trn s a l thuyt trn n v chiu di (N):

N = 5.54(ye/W1/2)2 X 1000/L

Trong :

Ve: th tch mi gii liW1/2: chiu rng mi ti mt na chiu cao mi.L: chiu cao m

Hiu qu c th c ci thin bng cch gim kch thc ht, tuy nhin kch thc ht nh to tr lc ln v tc dng gim, dn n thi gian tin hnh ko di.

ng u ca lp m v ht nhi s nh hng n ng u ca dng chy qua ct v do s nh hng n hnh dng v rng ca mi. Thit b sc k gel vi ng u cao lun cho mi gii li hp.

Loi sc k gel vi kch tht ht nh to iu kin cho cc phn t d dng khuch tn vo v ra khi ht, gip gim thi gian t cn bng gia pha ng v pha tnh, v vy ci thin c gii li do gim chiu rng mi.

S pha long mu l khng th trnh khi bi v khi mu i qua ct th qu trnh khuch tn xy ra. gim ti thiu vic pha long mu th s dng th tch mu ti a trong gii hn khong cch c th phn tch. Nu khng c vng gin rng th th tch ti a ca mu c th ln bng th tch phn tch:

VSep = VeB - VeA

Tuy nhin, do dng khuch tn xoy, s khng cn bng gia pha tnh v pha ng, khuch tn dc theo lp m, nn lun c vng gin rng. V vy th tch mu lun nh hn th tch phn tch.

Th tch mu nh

Th tch mu ti a c th phn tch nu khng c vng gin rng

Th tch mu ti a c th phn tch trong iu kin thc nghim

Hnh II.8. ng cong gii li cho th tch mu khc nhau

PHN LOI GEL

Vic tch trong sc k gel ty vo c im ca l rng trong h mng khng gian ba chiu. Ht gel phi c kch c ging nhau, c tnh tr v mt ha hc, bn v mt c hc, cc l rng trong ht gel phi c hnh dng ng nht.

Mi loi gel c kh nng xc nh trong vic tch mt loi trng lng phn t, ty thuc vo kch thc ca l rng trong ht gel. Cc loi gel thng mi thng khng chu c tnh acid hay base qu mnh, l rng khng qu ln v s lm gim bn c hc.

C hai loi nguyn liu chnh ch to gel l xerogel v aerogel. Xerogel l loi gel c in, l mt polymer c to mng ngang, khi cho tip xc vi dung mi, gel s trng n to thnh mng gel vi cc l rng tng i mm. Trong h gel ny cc l rng l khong trng gia nhng chui dy trong h thng mng. Nu dung mi c loi khi h thng mng, cu trc gel b xp , nu cho dung mi tr li, i khi c th khi phc li h mng .

Ngc li aerogel l nguyn liu rn chc v khng phi l gel, th d nh thy tinh c l rng hoc silica c l rng. l nguyn liu rn c l rng, dung mi c th chui vo cc l rng ny, nu dung mi c loi khi h thng mng, cu trc gel khng b xp .

Khi la chn loi gel thch hp cn xem xt hai yu t:

Mc tiu ca th nghim (phn tch phn gii cao hay tch thnh tng nhm

hp cht).

Khi lng phn t ca protein mc tiu v cht bn.

Trn th trng c 5 nhm thng dng l Superdex, Sephacryl, Superose v Sephadex v Sephadex LH-20

Superdex

Superdex l la chn cho gii li cao, thi gian ngn v thu h cao

T phng th nghim n qui m cng nghip, Superdex l loi gel c la chn u tin cho gii li cao vi thi gian ngn v thu hi tt.

Superdex l mt loi composite da trn lin kt ngang gia cc ht rng agarose n mng dextran bng lin kt cng ha tr. N c bn vt l v ha hc cao bi mng agarose lin kt ngang v c tnh cht lc gel tt do chui dextran. bn c hc ca Superdex cho php nhng dung mi gii ly c nht cao nh urea 8M vn chy qua c. Loi gel ny c th chu ng tc dng cao trong sut qu trnh n nh hay lc ra ct. Tnh bn gip loi Superdex ph hp cho s dng trong cng nghip vi tc dng cao, nhanh v hiu qu lm sch ti ch. Nhng tng tc gia protein v gel Superdex c b qua khi s dng dung mi vi cng ion trong khong 0,15 M n 1,5M.

Hnh III.1. Mng li lin kt trong Superdex

K.

1.00 -I

0.75 -

0.50 -

0.25 -

Hnh III.2. ng cong chn lc cho Superdex (13 ^m) v Superdex prep grade (34 rn)

Superdex c sn xut vi hai loi kch thc ht (13 v 34 ^m) vi 4 chn lc khc nhau (Superdex Peptide, Superdex 30, Superdex 75 v Superdex 200).

S dng ct c nhi sn ht kch thc 13 ^m ca Superdex Peptide, Superdex 30, Superdex 200 cho phn tch phn gii cao vi th tch mu nh.SC K GEL - NG DNG

SAC K GEL - NG DUNG

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SAC KI GEL - UNG DUNG

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S dng ct c nhi sn ht kch thc 34 ^m ca Superdex prep grade cho qui m cng nghip.

Khi khng bit khi lng ca protein quan tm th nn bt u vi Superdex 200. Superdex 200 hay Superdex 200 prep grade c bit thch hp cho vic phn tch khng th n dng t imer v t cht bn c khi lng phn t thp.

Superdex Peptide hay Superdex 30 prep grade dnh cho vic phn tch peptides, oligonucleotides v phn t protein nh (Mr < 10000).

Bng III.1. Thng s k thut ca cc loi ct nhi sn gel Superdex

ProductT

Fractionation range, Mr (globular proteins]

Sample loading capacity*

Maximum operating back pressure

Rec Dm mended operating flow T

Superdex Peptide PC 3.2/30

1x102-7x10*

25-250 pi

1.8 MPa. 18 bar, 260 psi