Thu cn, ha vi m phosphate thnh dchT phn vi ncLy tm 5000 v/pht, 4oC/10 pht

Trn quDch enzyme thTa vi acetonTinh sch bng sc k lc gelLy tm 5000 v/pht, 4oC/10 pht, lc thLc qua mng 250nmin diHn hpXc nh hot tnh protease, acid amin. Xc nh hm lng protein

Xc nh mcht kh

Xc nh hot tnh protease, acid amin. Xc nh hm lng protein

Xc nh hot tnh protease. Xc nh hm lng protein

1. Xc nh m ca trn qu1.1. nh nghaHm lng m ca bt l phm trm ca khi lng mt i sau khi sy 102oC cho n khi khi lng bt khng thay i.1.2. Nguyn tc:Sy kh mu cho n khi lng khng i 102oC 2oC trong khong 4h. Tip tc sy kh cho n khi no khi lng gia 2 ln sy khng vt qu 0.5mg. 1.3. Ti liu tham kho- International IDF Standard 26A:1993- FAO FNP 14/7, p.205-1986 Xc nh hm lng m1.4. Thit b- T sy c thng gi tt- a cn c np y. - Bnh ht m (cha cht lm kh tt nh Sillicagel)- Cn phn tch, chnh xc 0.001g1.5. Tin hnh- Sy kh da v np y da. (khng y np ln da). V lm ngui trong bnh ht m - Cn da v np v ghi khi lng l (mo). Cn khong 2.5g 0.5g mu cho vo da, y np v cn li da v ghi khi lng (m1).- t da v np vo t sy (khng y np ln da) v sy trong 2 gi 102oC 2oC- Sau khi sy ly np y da li v chuyn qua bnh ht m lm ngui v cn ghi khi lng l (m2).- Tip tc sy mu (phi m np) trong khong 1 gi 102oC 2oC- Thc hin li bc 5.4 v cn li khi lng (m2)- Lp li bc 5.5 v 5.6 cho n khi no s chnh lch gia 2 ln cn khng qu 0.5mg1.6. Tnh kt quHm lng m c tnh nh sau:

Trong :1. m0: khi lng np v da (g)1. m1: khi lng np + da + mu (g)1. m2: khi lng np + da + mu sau khi sy kh (g)2. T phn vi ncT l l 13% (13g trn kh trong 100ml nc) trong 10 ngy3. Xc nh hm lng protein S dng phng php Bradford3.1. Nguyn tcDa vo phn ng mu ca protein vi xanh Coomassie (Coomasie Brilliant Blue G-250) v kh nng hp ph nh sng bc sng 595nm. Trong mi trng acid Coomasie Brilliant Blue G-250 lin kt vi protein, tng tc vi c nhm k nc v nhm mang in tch dng trn phn t protein lm dung dch c mu xanh. Cng mu t l vi nng protein trong dung dch. Da vo 9dng chun suy ra lng protein trong mu3.2. Dng c v ha cht Dng c: My lc My o mt quang ng nghim: 20 ng Becher 100ml, 500ml Pipet 1ml, 5ml, 10ml Ca nha 5 lt Cn Giy lc Ha cht:1. Dung dch Albumin chun 1mg/1ml1. Thuc th Bradford1. Dung dch NaCl 0.15M3.3. Thc hin Dng ng chunChun b dung dch mu Albumin chun 1.5mg/ml.Cch pha dung dch Albumin: cn chnh xc 0.075g pha trong 50ml NaCl 0.15M, lc u cho tan ht v gi 20oC. Khi s dng pha long c nng Albumin chun 0.25mg/ml, 0.5mg/ml, 0.75mg/ml, 1mg/ml v 1.5mg/ml.Pha thuc th Bradford: + Cn 0,1g Coomassie Brilliant Blue vo 50ml Ethanol 96o, pha long vi nc ct thnh 5000ml: dung dch (1)+ 100ml acid phosphoric 85%, pha long trong nc ct thnh 500ml: dung dch (2)+ Pha dung dch (1) + dung dch (2) lc ly dch trongDng ng chun bng cch o OD ti cc nng khc nhau nh trn ca albumin, trn 1ml albumin vi 5ml dd Bradford. Lc u v yn hn hp phn ng ti thiu trong vng 5 pht ( ti a khng qu 1 gi) nhit phng trc khi o bng my quang ph bc sng 595nm. Thc hin trn mung th nghim: 1ml dd protein + 5ml dd Bradford ng i chng: 1ml NaCl 0.15M + 5ml dd Bradford o hp thu bc sng 595nm v ghi nhn mt quang (mt quang phi nm trong khong ca ng chun).3.4. Tnh kt qu:Tng hm lng protein trong M(g) protein th mg protein= b*10-3*L*Mb: nng protein suy ra t ng chun (mg/ml)L: h s pha longM: khi lng ch phm enzyme th (g)4. Xc nh hot tnh proteasePhng php Anson ci tin4.1. Nguyn lCasein b phn gii trong mi trng kim di tc dng ca protease to sn phm l cc on peptide ngn ha tan trong tricloroacetic acid (TCA), xc nh lng tyrosine v tryptophan ha tan bi thuc th Folin.4.2. Dng c v ha cht Dng c1. My lc1. My o mt quang1. Bp n nhit1. Bnh tam gic250ml.1. Bnh nh mc 10 ml, 100 ml, 500ml1. ng nghim1. Pipet 1 ml, 5 ml, 10 ml Ha cht Dung dch Na2HPO41/15M: ha tan 5,9690gNa2HPO4.12H2O trong nc va 250 ml Dung dch KH2PO41/15M : ha tan 0,9072 g KH2PO4trong nc va 100 ml Dung dch m Sorensen pH 7,6: trn chung 88,5 ml dung dch Na2HPO41/15M vi 11,5 ml dung dch KH2PO41/15M n khi pH t 7,6. Dung dch casein 1%:pha 1g Casein trong 100 ml dung dch m Sorensen Dung dch TCA 5%: ha tan 5 g TCA trong nc cho 100 ml Dung dich NaOH 0,5N: ha tan 10 g NaOH trong nc cho 500 ml. Thuc th Folin( pha long vi nc ct theo t l 1:4). (bn trong mt acid nn phi tin hnh th nghim nhanh) Dung dich HCl 0,2N: trn 4,25 ml HCl m c vi nc cho 250 ml Dung dch Tyrosine 20 M/l : khuy nghin 1,8119 g Tyrosin trong dung dch HCl 0,2N va 500 ml Dung dch Tyrosin chun 1 M/ltrong dung dch HCl 0,2N: pha long 5 ml dung dch Tyrosine 20 M/l trong dung dch HCl 0,2N thnh 100 ml4.3. Chun b ng chun Chun b dung dch tyrosinchun-T dung dch tyrosin chun1 M/l, xy dng ng chun vi dung dch tyrosin c nng t 0,2 - 1,0 M/l .Dung dch ha chtng nghim

123456

Dung dch tyrosine chun (ml)0,20,40,60,81,00

Lng tyrosine tng ng (M)0,20,40,60,81,00

Dung dch HCl 0,2N (ml)4,84,64,44,24,05,0

Dung dch NaOH 0,5N (ml)101010101010

Thuc th Folin (ml)333333

Lc mnh, sau 10 pht o OD bc sng 660nm

ng s 6 l ng kim chng (KC), cc ng cn li l ng th nghim (TN). V ng chun tyrosine tng quan tng quan gia lng tyrosine (M) v OD (OD = ODTN- ODKC).4.4. Phng php tin hnh Chit muMu cn chnh xc 10g ( i vi mu rn) hoc 10 ml (i vi mu lng) cho vo becher, nh mc va 100ml bng nc ct em lc trn my lc trong vng 15 pht. em ly tm lnh vi 4000 vng/ pht trong 10 pht, thu dch, b b (i vi mu lng khng cn ly tm, ch lc ng nht mu). Tin hnh Ly 6 ng nghim sch, kh, tin hnh lm 3 ng th tht, 3 ng th khngDung dch ha chtng nghim

Th thtTh khng

Dung dch casein 1% (ml)55

Dung dch TCA 5% (ml)010

Dung dch enzyme mu (ml)11

Lc u v gi 35,5oC trong 20 pht

Dung dch TCA 5% (ml)100

yn 30 pht, lc ly dch bn di

Ly 2 ng nghim mi khc,cho vo ng th nht 5ml dch lc ca ng th tht v cho vo ng th hai 5ml dch lc ca ng th khng.Tip tc thm vo mi ng 10ml NaOH 0,5N v 3ml thuc th Folin, lc mnh, sau 10 pht o OD bc sng 660nm. OD = ODTT ODTK, sau da vo th chun suy ra c M tyrosine.4.5. Tnh kt qunh ngha n v Anson: mt n v Anson l lng enzyme ti thiu trong iu kin th nghim ( 35,5oC: pH 7,6 ) thy phn casein trong 1 pht to thnh sn phm ha tan trong TCA, phn ng vi thuc th Folin cho ta hp thu OD bc sng 660nm tng ng vi 1 M tyrosine trong ng chun.H Protease = Vi:H Protease : hot enzyme protease ( UI/g)V1: th tch pha long ban u : 100 mlV : tng th tch hn hp trong ng th tht hoc ng th khng(ml)v : th tch dch lc em phn tch (ml)t: thi gian thy phn (pht)m : khi lng mu enzyme em i xc nh hot tnh (g)L: pha long enzymeM tyrosine: lng M tyrosine trong v (ml) suy ra t ng chun5. Xc nh hm lng acid amin bng ninhydrin5.1. Nguyn tcDi tc dng ca ninhhydrin, acid amine b oxi ha chuyn thnh amino acid ri n ammoniac v carbonic. Amino acid b gim bt 1 nguyn t C tr thnh dng aldehyde. Amino acid c khi lng phn t nh hn v ammoniac to thnh s oxi ha ninhydrin to nn phc hp c mu xanh tm, cng mu tng ng vi gi tr nit amin ca amino acid.5.2. Dng c v ha cht: Dng c: Bnh nh mc 10ml : 10 bnh Pipet 1ml, 5ml ng nghim Ni My so mu Ha cht: Dung dch Ninhydrin : ha tan 2g Ninhydrin trong 50ml dung dch ethanol 50% Dung dch m citrate 0.4M, pH = 5 Dung dch ethanol 50% Dung dch chun leucin c nng (?)5.3. Dng ng chun Leucin:1. T dung dch chun leucin (?) pha long thnh cc dung dch chun 2 g/ml, 3 g/ml, 4 g/ml v 5 g/ml1. Chun b 6 ng nghim trong c 1 ng i chng1. Cho ln lt vo 5 ng nghim 5 loi dung dch chun leucin, mi ng 0.5ml, ring ng i chng thay bng 0.5ml nc ct.1. Cho thm 1.5ml dung dch m citrate 0.4M, pH = 5 v 2ml dung dch ninhydrin vo mi ng nghim.1. Lc u ng nghim v t vo ni nhit un si 30 pht.1. Cho thm ethanol 50% vo n 10ml mi ng nghim.1. Lc u v o hp thu bc song 570nm.1. Dng ng chun leucin.5.4. Tin hnh:1. Ly 0.5ml dung dch sau ly tm vo ng nghim sch, kh, cho thm 1.5ml dung dch m citrate 0.4M, pH =5 v 2ml dung dch ninhydrin.1. Lc u ng nghim v t vo ni nhit un si 30 pht.1. Cho thm ethanol 50% vo n 10ml mi ng nghim.1. Lc u v o hp thu bc sng 570nm.1. Ghi nhn kt qu.1. Tnh kt qu:6. Ta enzyme bng acetone1. Kho st t l dch trn qu-dung mi (1:1, 1:2, 1:3, 1:4, 1:5)1. Thi gian ta (30, 60, 90, 120, 150 pht)7. Tinh sch bng sc k lc gel8. in di8.1. Chun b mu Mu kh hoc dung dich c hoa tan trc tip trong dung dich mi pha mu hoc ti l 1:1 v th tich gia mu va dung mi pha mu sao cho co nng protein khoang 5mg/ml, un si cach thuy 5 phut, ngui mi cho vao cac ging cua gel.Pha thang protein chun phn t lng thp (14,4kDa-94kDa)( nu co).8.2. Chun b gel phn tch 1. To khun bn gel:Gel polyacrylamide thng t ng, ch gel cn s dng cc tm thy tinh c kt cu chuyn dng lm khun.Ra sch cc tm thy tinh k bng nc sch, kh ri lau li bng ethanol. Rp cc tm thy tinh theo hng dn ca hng sn xut sao cho to c khong h gia hai tm thy tinh c bt kn ba pha khng lm chy nguyn liu to gel khi rt vo. Trc tin t tm thy tinh nguyn, t cc thanh cch dc theo mp tm thy tinh , ri t tm thy tinh c "tai" ln trn sao cho cc cnh ca hai tm thy tinh trng kht nhau, dn cc mp bn v khe h y bn gel bng bng keo ri dng kp kp cc tm thy tinh (kp trc cc tm thy tinh n nh, dn tng cnh ri thay kp).

Hnh 3.2 S khun bn gel polyacrylamidem gel phn tach1.5 ml

H2O2.5 ml

Dung dich Acrylamide2 ml

Amonium persulfate20 l

Lc nhe cho tan ht

TEMED4l

- Khi cho TEMED lc nhe cho tan ht sau o dung pipette hoc micropipette bm dung dich gel phn tach vao khoang hep cua hai tm kinh (khun) ma khng tao bot khi sau cho mc dung dich gel cao 8cm, nhe nhang t mt lp nc ct trn lp gel, ch cho gel cng.Chun b gel gom :m cua gel gom0.625 ml

Nc ct1,525 ml

Dung dich Acrylamide0,325 ml

Amonium persulfate12.5 l

Lc nhe cho tan ht

TEMED2 l

Sau khi gel phn tach a cng hoan toan (15-30 phut), hut ht nc bn trn sau o lp gel gom vao khun bng cach dung pipette hoc micropipette, ng thi a lc vao gel tao cac ging, sau khi gel gn cng ly lc ra nhe nhe ri lp khun gel vao b in di, cho dung dich in di vao hai bung in di, bm t 5 -15l mu ( muc 2) vao ging theo th t, y hp in di, ong in, tin hanh chay in n inh dong (2mA/ging), thng vi in th khoang 100 V-120 v va cng dong in khoang 30mA, khoang 1 gi sau khi vach mau chi thi xung gn n ay cua gel tt in va ly gel ra nhum. Cc bc tin hnh sau khi in di :- Ly gel ra khoi tm kinh.- Ngm gel nay trong ia petri co cha thuc nhum protein, lay ng nhe cho n khi thuc nhum thm vao gel, thi gian ngm (30 pht - 1 gi).- Chuyn gel sang ngm trong dung dich ra mau, thay dung dich ra mi vai ln cho n khi gel co mau trong sut, luc nay se thy xut hin nhng vach xanh l trn gel.8.3. Kt lunPhn tch kt qu, chp hnh gel v xc nh trng lng phn t phn tch kt quo khong cch di chuyn ca cc band protein t gel phn tch ti cc band protein v khong cch di chuyn t gel phn tch ti vch mu cui cng