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vn Tnh tan ca protein chu nh hng mnh ca pH v nng mui trong dung dch.

nh hng ca pH
Protein tan t nht pI do tng tc tnh in ca phn t vi dung mi l thp nht
Solubility pH

pI

nh hng ca nng mui

Salting in l hin tng tng Salting in ho tan khi tng nng mui; Salting out Salting out l hin tng gim ho tan khi tng nng mui; Salt Kt ta bng mui ammon l phng php thng dng da trn hin tng salting out. Ti liu chia s ti: wWw.SinhHoc.edu.vnconcentration
Solubility

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Tinh ch Protein
Nhng phng php thng dng trong tinh ch protein:
Sc k lc gel; Sc k trao i ion; Sc k i lc; Sc k tng tc k nc;

Xc nh sch v kch thc protein bng in di bin tnh trn gel polyacrylamide (SDS- PAGE)
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Sc k lc gel (Ry phn t)

S phn tch protein da trn kch thc phn t: Cc ht gel cu to t polymer ca dextran (sephadex), agarose (Sepharose), polyacrylamide (Sephacryl hoc BioGel P). Mi ht gel u c nhng mt li to hay nh ph thuc vo lo gel. Trong qu trnh sc k, cc cht phn t ln s ch di chuyn pha lng pha ngoi ht gel, v s nhanh chng rt ra khi ct. Trong khi , cc cht c kch thc nh hn s chui qua cc mt lay vo bn trong ht gel v s vi mc khc nhau tu theo khi lng v hnh dng ca chng, v s ra khi ct gel chm. Phn t cng nh cng phi len lch nhiu trong ht gel, v cng ra chm. Do vy c th coi vic tch cc cht bng gel Sephadex nh mt qu trnh sng lc (ry) phn t. Cc phn t c phn b th ng gia th tch k (th tch bn ngoi ht gel- V0) v th tch bn trong ht gel (Vi), ph thuc vo kh nng chui c vo bn trong ht gel ca chng. Nu tng th tch ca ct l Vt, th: Vt = Vo + Vi
hp th UV
Protein ln Protein nh hn

V0

Vi

Vt

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Sc k trao i ion
Sc k trao i ion (ion exchange chromatography) s dng tnh cht tnh in ca protein cn tch. Trong qu trnh sc k, cc cht phn t ln tch in s c c nh qua cc lin kt cng ha tr ln mt cht gi khng tan nh cellulose hoc polyacrylamide. Protein vi in tch tri du s gn vi gi th trong khi cc protein khc b ra tri khi ct. Sau khi ra ht tp cht, protein gn vi gi th c chit rt bng lc ion cao (mui nng cao), hoc bng cc tc nhn khc nh thay i pH
protein B protein B Protein B Protein A Protein B

Nng mui tng

Na+

SO3+ --CM

ProteinA

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hp th UV

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sc k trao i ion
Cc c tnh tnh in ca mt protein xc nh loi nha trao i ion c tc dng ph hp. V nguyn tc l: Protein tch in dng nu dung dch c pH < pI, khi n s gn vi nha tch in m, hay cn gi l cationit (cation exchanger); Protein tch in m nu dung dch c pH > pI, khi n s gn vi nha tch in dng, hay cn gi l anionit (anion exchanger) Lu trn thc t b mt protein c nhng vng c in tch khng phn t. khc vi tng in tch ca ton bphn cc

B mt+ + - protein+ --

khng phn cc

V d v cationit gm: carboxymethyl (CM) v sulfopropyl (SP) V d v anionit gm: diethylaminoethyl (DEAE), quaternary amine (QAE)

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Sc k I lc
Sc k i lc (affinity chromatography) s dng i lc gia cu t gn (ligand) v protein cn tch. Cu t gn thng c c nh qua cc lin kt cng ho tr gi th khng tan nh cellulose hoc polyacryl-amide. Khi tin hnh sc k, protein cn tch s gn vi gi th trong khi cc protein khc s ra tri ra khi ct. Sau khi ra sch tp cht, protein cn tch c phn hp ph bng cc cht cnh tranh nh cu t t do, hay bng cc cht c kh nng ph v tng tc. Trong s cc v d v tng tc protein- cu t c th k n tng tc khng nguyn- khng th, hay tng tc gia Ni+ v nhn poly-histidine.
hp th UV

protein ligand

Nng cu t t do

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Tng tc k nc
Sc k tng tc k nc (hydrophobic interaction chromatography) da trn nhng mng k nc trn protein v tng tc ca chng vi nha k nc. V d, phenyl sepharose l mt loi nha k nc mnh thu c khi gn cng ho tr nhm phenyl vi gi th agarose. Trong qu trnh sc k, nhng phn t protein gn vi nhn phenyl nh tng tc k nc. Nhng tng tc ny l mnh nht trong dung dch mui nng cao. Do vy quy trnh thng thng ca phng php l trc tin gn protein iu kin nng mui cao, sau chit rt protein gn bng gradient mui ngc t nng cao xung thp.
khng phn cc
hp th UV

B mt + + - protein +

Nng mui gim

--

khng phn cc

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Sc k giy

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