I.

Phng php xc nh hot tnh enzyme carboxymethyl cellulase (CMCase) a. Nguyn tc: Phng php ny da vo s thy gii CMC bi enzyme carboxymethyl cellulase pH = 5 v 40oC. Sau p hn ng thy phn s to ra mt lng ng kh, ng kh s phn ng vi acid 3,5 dinitrosalicylic (DNS), mu san phn ng c xc nh bng o mt quang bc sng 540nm. nh ngha: Mt n v hot tnh CMCase l lng enzyme cn thit gii phng 1mol/ pht di cc iu kin thc nghim. b. Ha cht: - Dung dch c cht (1%W/V CMC): cn chnh xc 1g CMC, ha tan trong 80 ml dung dch m Na acetate 50 mM, pH 5, chuyn vo bnh nh mc 100 ml, thm nc n vch v lc u. - Dung dch acid 3,5 dinitrosalicylic: cn 10g DNS cho vo becher 1000 ml, thm khong 400 ml nc ct, t becher trong chu nc 80oC v khuy u. Thm dung dch NaOH (16g NaOH trong 150ml nc ct), t t vo d ung dch DNS, khuy 180oC. Tip tc thm 300g potassium sodium tartrate tetrhydate vo d ung dch DNS v tip tc khuy nhit 80oC. Lm lnh dung dch DNS n nhit phng. Chuyn duch dch vo bnh nh mc 1000ml, thm nc ct n vch, lc u. Bo qun dung dch trong chai mu nu c np. - Dung dch lactose: ha tan 0.12 g lactose monohydrate vi 80ml nc ct v chuyn vo bnh nh mc 100ml, thm nc ct n vch, lc u. - Dung dch DNS lactose: trn u 150ml DNS vi 50 ml dung dch lactose. - Dung dch enzyme: Pha long dung dch enzyme vi dung dch m Na acetate 50mM, pH 5 n pha long thch hp. c. Dng ng glucose chun - Hon tan 100mg glucose monohydrate vi 50ml nc ct v chuyn vo bnh nh mc 100ml, thm nc ct n vch v lc u - Xy dng ng glucose chun theo bng sau: ng s 0 1 2 3 4 5 6 Nng glucose (mg/ml) 0 0.1 0.2 0.3 0.4 0.5 0.6 - ml dd glucose (1mg/ml) 0 0.1 0.2 0.3 0.4 0.5 0.6 - ml dd CMA 1% 1 1 1 1 1 1 1 -ml dd DNS lactose 2 2 2 2 2 2 2 -ml nc ct 1 0.9 0.8 0.7 0.6 0.5 0.4 - Lc u cc ng nghim ny, em un si cch thy trong 15 pht. Lm lnh n nhit phng trong mt chu nc mt. So mu trn my quang ph bc sng 540 nm d. Tin hnh phn ng - Ht 1ml dung dch enzyme cho vo ng nghim th tht, thay 1ml dung dch enzyme bng 1ml dung dch Na- acetate 50mM, pH 5 i vi ng th khng. t vo b n nhit 40oC/5phu1t. ng thi ta cng t l cha dung dch CMC 1% 40oC/5 pht.

Thm 1ml dung dch CMC 1% vo ng nghim cha enzyme v lc u. Cho phn ng 40oC trong thi gian 10 pht. Thm 2ml dung dch DNS lactose, lc u ngng phn ng enzyme. em un si cch thy trong 15 pht. Lm lnh n nhit phng trong mt chu nc mt. So mu bc sng 540nm da theo ng glucose chun. e. Tnh ton Gi tr F = (0.1/AG0.1 + CMCase

Sc k lc gel Khi qut: Sc l thut dng phn tch cc hp phn khc nhau ca hn hp, da trn s di chuyn hc nhau trong pha ng ca cc cht ha tan trong hn hp. Qu trnh chung ca sc k - Ct sc k chun b vi mu cn phn tch, gi th c cn bng vi dung dch m. - Mu phn t sinh hc c ha vo dung dch m v c a qua ct - Dng dung dch m c bm qua ct, mu s c y ra khi ct theo tng giai on. Sc k lc gel s dng gi th l cc ht gel, s phn tch da trn ch thc phn t. Nguyn tc: S tch bit cc phn t protein da trn s khc nhau v ch thc, khi hn hp protein di chuyn dc theo ct ch a cc ht gel c ch thc l quy nh theo c ch thm thu, dn n s khuych tn chn lc. Cu to: - Pha tnh l mt ct ch a cc ht gel. - Pha ng ch a dung mi chuyn ng. - Mt u d c gn v o im cui ca ct. - c lm t polime hay cacbonhidrate c tnh tr, hng ha tan, hng phn ng vi protein - B mt cc ht gel c cc l vi ch thc xc nh. - c lm t polime hay cacbonhidrate c tnh tr, hng ha tan, hng phn ng vi protein - B mt cc ht gel c cc l vi ch thc xc nh.
BO CO TH NGHIM PROTEIN ENZYME GVHD: TS. HUNH NGC OANH SVTH: NHM 3 BUI SNG 52

Hnh 8. Minh ha phng php lc gel Cc bc tin hnh th nghim: - Nhi ht gel vo ct. Dng ng ht cc ht gel ch a trong dung dch m bo v nhi t t vo ct m bo ct gel to thnh khng ch a bt. - Dng nc ct ra sch m bo v gel, sau cho m Tris-HCl 0.05M qua ct trong 1 gi . Kt thc dng nt bt ct gel trn mt gel vn cn khong 1cm

chiu cao dung dch m. - Tin hnh cho dung dch m vo ct gel v g nt bt ra tin hnh qu trnh chy sc k lc gel. Trong qu trnh chy phi cho m vo ct gel m bo trn mt gel trong ct vn cn khong 1cm chiu cao dung dch m. - Khi qu trnh chy tin hnh thu mu ra khi ct cho vo ng nghim. Mi ng nghim thu khong 3ml (hay 50 git). - Khi thu c 3 ng ch a mu th cho 2ml dch enzyme th vo ct gel. Qu trnh tip tc din ra v phi m bo trn mt gel lun c khong 1cm dung dch (dung dch enzyme hay m). - Cc mu trong ng nghim c em i o OD bc sng 540nm xc nh mu ch a enzyme tinh. - Qu trnh chy sc k kt thc khi c 3 mu cc ng nghim k nhau m ch s OD hng i.
BO CO TH NGHIM PROTEIN ENZYME GVHD: TS. HUNH NGC OANH SVTH: NHM 3 BUI SNG 53

2.2.2.4. in di Nguyn tc ca k thut in di in di (Electrophoresis) l k thut phn tch cc thnh phn trong hn hp da vo vn tc chuyn ng khc nhau ca cc ion trong in tr ng. Vn tc chuyn ng ca cc cation ti cathode hay ca cc anion ti anode ph thuc vo lc tc ng ca in tr ng ln cc ion mang in v ph thuc vo cc lc tng tc gia cc phn t, gia phn t vi mi tr ng, m ch yu l lc ma st v lc tng tc tnh in. Rt nhiu hp cht sinh hc ch a cc nhm c kh nng ion ha v d nh axit amin, peptit, protein, nucleotit, cacbohydrat v.v. trong dung dch chng c th tn ti dng tch in, hoc l cation hoc anion. a s cc phn t sinh hc l cc cht in ly lng cc. Du in tch ca phn t ph thuc vo pH. Mt khc c nhiu cht mc d mang in tch phn t gn ging nhau hay thm ch c in tch bng nhau, nhng do hc nhau v trng lng phn t, nn c gi tr tng quan t l gia in tch phn t v trng lng phn t (q/M) khc nhau. V vy m vn tc chuyn ng trong in tr ng ca mi phn t tch in l khng ging nhau. chnh l c s ca k thut in di, phn tch ring cc cu t trong in tr ng do s khc bit v in tch v phn t lng. Tin hnh: - Mu dung dch c ha tan trc tip trong dung mi pha mu t l 1:1 v th tch gia mu v dung mi pha mu sao cho c nng protein khong 1 mg/ml. un si cch thy 5 pht, ngui mi cho vo cc ging ca gel - Chun b gel phn tch - To khun bn gel Gel polyacrylamide th ng t ng ch gel cn s dng cc tm thy tinh cho c kt cu chuyn dng k lm khun. Ra sch cc tm thy tinh k bng nc sch, kh ri lau li bng ethanol. Tp cc tm thy tinh c bt n ba pha khng lm chy nguyn liu to gel khi rt vo.

Trc tin t tm thy tinh nguyn, t cc thanh cch dcoj theo mp tm thy tinh ,
BO CO TH NGHIM PROTEIN ENZYME GVHD: TS. HUNH NGC OANH SVTH: NHM 3 BUI SNG 54

ri t tm thy tinh c tai ln trn sao cho cc cnh ca hai tm thy tinh ny trng kht vi nhau, dn cc mp bn v khe h y bn gel bng bng eo ri dng kp kp cc tm thy tinh (kp trc cc tm thy tinh n dnh, dn tng cnh ri thay kp). Bng 2. Cng th c ca gel phn tch m gel phn tch 0.5ml H2O 1.18ml Dung dch Acrylamide 0.3ml SDS (10%) 20l Amonium persulfate 1l Lc nh cho tan ht TEMED 4l - Khi cho TEMED lc nh cho tan ht sau d dng pipette hoc micropipette bm dung dch gel phn tch vo khong hp ca hai tm knh (khun) m khng to bt kh sao cho m c dung dch gel cao 8 cm, nh nh ng t mt lp nc ct trn lp gel, cho cho gel c ng. - Chun b gel gom: Bng 3. Cng th c ca gel gom m gel gom 0.5ml H2O 1.18ml Dung dch Acrylamide 0.3ml SDS (10%) 20l
BO CO TH NGHIM PROTEIN ENZYME GVHD: TS. HUNH NGC OANH SVTH: NHM 3 BUI SNG 55

Amonium persulfate 1l Lc nh cho tan ht TEMED 1l -