chuong 5.pphshd.hkiii

7/25/2019 Chuong 5.Pphshd.hkiii

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TRNG I HC TN C THNG

KHOA KHOA HC NG DNG

B mn Cng ngh Sinh hc

- 603029

CC PHNG PHP HA SHIN I

Ging vin: Trng Th D

Email: truongthidieuhien


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1. L thuyt tinh2. K thut in d

C5. Tinh sch Protein K

603029C5. Tinh sch protein & KT in di


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1. Tinh sch Protein



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Protein purification is a series of

processes intended to isolate one ora few proteins from a complexmixture, usually cells, tissues orwhole organisms.Protein purificationis vital for the

characterization of the function,structure and interactions of the

protein of interest.

1. Tinh sch Protein



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The purification process may separate the

protein and non-protein parts of the mixture,and finally separate the desired protein fromall other proteins.Separation of one protein from all others istypically the most laborious aspect of protein

purification.Separation steps usually exploit differencesin protein size, physico-chemical properties,

binding affinityandbiological activity.

1. Tinh sch Protein



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1. Tinh sch Protein



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1. Tinh sch Protein



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1. Tinh sch Protein



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1. Tinh sch Protein



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Da trn chuyn ng ca cc phn ttch

introng dungdchkhic avointrng. K thut in di l k thut tchnhng hp cht ca mt hn hp nh intrng

Cc phnttrongin trng chuyn ngvi vn tckhc nhauph thucvo:

+intch+ Hnhdng+ Kchthc

2. K thut in di Protei



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Nguyntcstchhp cht bng indi Ion mangintch tridu sdichuyn

theo haihng ngc chiunhau => cchp chttch ring ra

Ion mang in tch cng du tuy dichuyn cng chiu nhau nhng do dichuyn vi vn tckhc nhau nncngtchrinhau

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ng Dng:- Tch ringnhng hp cht ca

mt hn hp- pdngchonhng hp cht

thucCarbonhydrate sau khi c tothnh ccdn xut

borate hay phosphate

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1. ng dng tch v phn tch DNA

2. La chn h thng m, nng gel, iukin chy (dng in, thi gian, nhit ...)thch hp

3. S dng EtBr (Ethidium bromide) v tia

UV pht hin cc band DNA trn gel.

2. KT in di Protein: tr

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2. KT in di Protein: tr


PP H


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2. KT in di Protein: trn gel P


in di trn gel polyacrylamide cSodium dodecyl sulfate (SDS-PAGE):tch protein da trn KLPT (MW)

Khi tch bng SDS-PAGE s di ngc xc nh khng phi do in tchca chui polypeptide, m do khilng phn t(MW).

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PP H


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Ha cht:

Dungdch mgel phn tch (Resolving Gel)Tris HCl 1.5M pH 8.8

Dungdch mgel gom (Stacking Gel) Tris-HCl 0.5 M pH 6.8

Dung dch m indi (Buffer) Tris-glycine pH 8.0 (5X)

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gel: chun b mt gel poliacrylamide kch thc 100x100x0.75mm 10%,7.5%, 5%.

Gel phn tch:chun b mt dungdchgel phn tch ctng thtch lthnhphn c bsung vo erlen theoth tsau:

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Thnh phn Vgel 10%, l Vgel 7.5%, l Vgel

1.Nc ct 1700 2435 2

2.Tris-HCl 1.5M pH8.8 1250 1250

3. 30%acrylamide 2000 1250

4.SDS 20% 25 25

5.Amonium persulfate 10% 25 25

6.TEMED 2.5 2.5

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gel:

Gel phn tch:- Gel phn tchccho vo khungel

sao chomcdungdchgel cao 7 cm (trnhto btkh trong khun gel). Sau,chomt lp nc ctln trnlpgelva qu trnh polimer haxyra.Chgelng

khong20-30 pht.

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gel:Gel gom:chun bdungdchgel gomtng thtch 2.5ml c thnhph

c bsung theoth tsauThnh phn Vgel 4%, l

1.Nc ct 1540

2.Tris-HCl 10.5M pH 6.8 625

3. 30%acrylamide 3354.SDS 20% 13

5.Amonium persulfate 10% 13

6.TEMED 2.5

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gel:Gel gom: Sau khi gel phn tchtrngngng

hon ton, ht ncbn trn.Tinhnhlpgel gom,ng thi a lcvo gel to cc ging mu (trnh to bt kh trongkhun gel). Sau khi gel trngngng, ly lcra, thob phn bt y cakhun gel.Lp

a gel vob in di. Cho dung dch mindi vo my.

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Chun b mu v chy in di:

-Xlmu:bintnh protein-Tinhnhchy indin nhdng:iu chnhdnginthch

- Sauindi,tinhnhnhumgelvidungdch nhum. Sau khtrnhnhum tinhnhgii nhum.

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in di SDS-PAGE 1 chiu ( 11D Gel)


Cc protein ctch theotrng lngphn

t cachng (MW)Thc hin indi ng thi2 muMuprotein cn khost (sample) cload

voging(well)Hn hpprotein chun(Protein ladder) dng

i chiu cload voging(well) utinhoc kbnging chaprotein cn khost.

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1 Dimension Gel1D Gel


Protein ladder

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in di SDS-PAGE 2 chiu ( 22D Gel)

Cc Protein ctch theo:+ Chiu th nht bng indi do imng in(Isoelectric focusing IEF).Protein cphn tch theointchcan.

+ Chiu thhaibngSDS-PAGE.Protein cphn tch theotrng lngphnt can (Molecular weight, MW)

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Tch theochiu th nht Tch theo chiu th hai

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in di min dch

K thut in thm Chuyncc thnhphn tchca mt hn hp muln trnmt

mngmngc tnhbn c hc=> tinhnh cc nghincukhc indi l nguyntc cn bn

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in di min dch

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in di min dch

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C5. n Tp

603029C1. SPTN v PPNC SPTN

1. Mc ch ca vic thc hin qu trnh tinh sch pro

2. in di protein: cc bc chnh thc hin KT in d

nguyn tc

3. in di 1 chiu: mc ch, nguyn tc, kt qu

4. in di 2 chiu: mc ch, nguyn tc, kt qu

5. Cho VD minh ha v vic ng dng KT in di prot

chuong 5.pphshd.hkiii

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