MC LC I/ T VN (1 TRANG) II/ LC KHO TI LIU

1. S lc v enzyme amylase: a. Amylase l g? b. Cc loi enzym amylase v cc yu t nh hng ti hot tnh amylase. * -amylase * -amylase 2. Phng php phn tch kim nh a. Phng php sc k trao i ion. . b. nh lng protein bng phng php Bradford c. Xc nh hot tnh amylase bng phng php Nelson
Nguyn tc: amylase thy phn tinh bt cho ra cc ng kh. Hot tnh

enzyme c xc nh da vo lng ng kh sinh ra thng qua phn ng trng gian vi thuc th Nelson Somogyi n v hot tnh enzyme
Cch tin hnh (ch cp mc - ni dung chnh khng lp li

phng php tin hnh chi tit trong gio trnh thc tp)
Cch xc nh hot tnh (cng thc)

d. Phn tch protein bng phng php in di trn gel polyacrylamide (SDS PAGE) Phng php ph bin xc nh trng lng phn t ca chui polypeptide l da vo dch chuyn ca phn t trong in di trn gel polyacrylamide (PAGC) vi s hin din ca cht ty sodium doecyl sulfate (SDS). Phng php ny tng ng vi st k lc gel im l kch thc phn t c xc nh da vo mc dch chuyn da vo h thng mng li ca gel. Tuy nhin phng php in di cc phn t nh dch chuyn nhanh hn cc phn t ln. Da vo ng chun protein vi trng lng phn t bit (ng chun c dng da trn mi tng quan gia dch chuyn v trng lng phn t, vi trc tung l Rf (t l gia
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 dch chuyn ca protein trn dch chuyn ca cc cht mu bromophenol blue) v trc honh l log10M. Khi SDS hin din nh l mt n phn t do vi nng cao s gy bin tnh hon ton cc protein v gy bin tnh cc protein, trung bnh l mt phn t SDS s bm hai gc amino acid. SDS bm vo protein l do tng tc ion, nn cc protein khng tch in th s khng b nh hng, mt d cc tng tc khng phn cc cng c vai tr nht nh. Do in tch SDS cao nn in tch protein xem nh khng ng k. Hng s bm ca protein biu hin s khc nhau ca phc hp SDS- protein c cng mt in tch m dc theo chiu di phn t. Ch cc protein hoc kim hoc acid to phc hp vi SDS theo kiu khng c trng phn nh mc tch din tht s ca protein. Mercaptoethanol (2-thioethanol) hay dithiothreitol cng c th c thm vo lm gim s ni disulfide. Cc phn t SDS mang in tch m s tng tc vi protein v bm dc theo chiu di phn t, ph v cu trc v hnh dng phn t, tch ri cc chui polypeptide v to cho protein c hnh dng tng i ging nhau cng mang in tch m. V vy di chuyn v cc dng trong in trng. Tc di chuyn ph thuc vo khi lng phn t ca chng; cc protein c khi lng phn t cng nh chy cng nhanh, ngc li cc protein c khi lng phn t cng ln chy cng chm, kt qu l phc hp SDS- protein c dch chuyn trong in trng t l nghch vi LogM ca protein. Khi in di kt thc, nhng protein trn gel s c nhn thy l mt dy cc band bng cch nhum vi bc hay mt cht nhum mu nh Coomassie blue. Nhng nh du phng x c th c pht hin bng cch t mt tm phim chp x quang ln ming gel, qu trnh ny gi l phng x t ghi. Nhng protein nh di chuyn nhanh trong gel, trong khi nhng protein ln pha u, gn v tr np mu. Tnh di ng ca phn ln cc chui polypeptide di nhng iu kin nh th t l vi khi lng ca chng. Tuy nhin, cng c vi protein giu carbohydrate v protein mng khng tun theo mi tng quan ny. SDS-PAGE c phn tch cao, cho kt qu nhanh v nhy cao. Ch vi lng nh khong 0.1ug (~2pmol) protein cho vch rt r khi nhum vi Coomassie blue v thm ch t hn (~0.02g) vn c th c pht hin khi nhum bng bc. Nhng protein chnh lch v khi lng khong 2% c th phn bit c bng SDS-PAGE. V vy, SDSPAGE gip chng ta nh gi kt qu ca qu trnh tinh sch. Vi nhng phn on u tin, kt qu l dy gm rt nhiu protein. Nhng qua mi bc tinh sch, s lng protein s gim v s c mt band ngy cng m. Vch tng ng vi protein mc tiu. So snh dch chuyn ca protein cha bit vi cc protein chun c phn t khi bit c th xc nh c c th xc nh c phn t khi ca protein cn tm vi sai s khong 10% gi tr tht. kt qu khng chnh xc i khi nhn c do s bt bnh thng trong tng tc gia SDS v protein hay do cu trc bt thng ca protein, s khc bit ln trong tch in ca protein hay do gc phi protein trong phn t nh gc ng chng hn. V vy cn phi ch khi s dng phng php ny. Bn cnh phng php trn cn c cc phng php khc dng nh gi tinh sch ca protein nh: e. nh gi kt qu tinh sch protein
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Sau mi bc tinh sch, nhng thng s sau cn c ghi nhn c th nh gi qu trnh tinh sch protein: - Protein tng s: . - Hot tnh tng: - Hot tnh ring: - Yield: - Mc tinh sch: Qua theo nghin cu kho nghim, ngi ta thy rng sau mi bc tinh sch, mc tinh sch tng ln trong khi yield li gim. Tht vy, vi k thut SDS-PAGE, nu ta np mt lng mu nht nh vo mi ging trn bn gel ng vi mt bc tinh sch, ta s thy s bng s gim t l vi mc tinh sch trong khi t l lng protein mc tiu vi tng s protein hin din s tng . V vy, nh gi qu trnh tinh sch phi da c vo 2 thng s mc tinh sch v yield. Mt qu trnh tinh sch tt s c mc tinh sch cao v ch s yield thp. Nu ch s yield cao cn mc tinh sch thp c ngha l cn nhiu tp nhim trong mu (nhiu protein ngoi protein mc tiu) v lm phc tp vic gii thch th nghim.

III/ PHNG TIN V PHNG PHP

1/ Phng tin: a/ Dng c v thit b: - H thng sc k lng - Bm - Cc thy tinh - Eppendoft - Giy lc Whatman no. 1 - Tip (xanh v vng) - My vortex - My trn Mixer - Bp cch thy.

- My khuy t. - My lc nhum mu. - My o quang ph - My sy gel. - ng nghim thy tinh - My o pH. - ng eppendorf, u cone, gng tay - Cuvette plastic - Hp ng gel - Pipetman - Dng c t eppendorf. - Beaker v cc dng c thy tinh phng th nghim. - My phn an sc k (fraction collector) - B in di gm khung in di, khung gel v b ngun. - Micropipette 20, 100, 200, 1000, 5000 l - ng eppendorf, u cone, gng tay b/ Ha cht: - Gel Uno Spere Q - m Tris-HCl 0,02M pH 8.0 - Comassie Blue G250 - Cn tuyt i - Acid phosphoric 85% - Bovine serum albumin (BSA) - Tinh bt (1% ) ha tan trong dung dch m maleic pH 6.0. - Dung dch m Maleic (11.6 g acid maleic ha tan trong 900 ml nc v chnh pH bng NaOH 2M v pH 6.0, thm 0.74 g CaCl2.H2O v chnh pH v 6.0 sau a th tch v 1 lt - 2% (w/v) Tris-baz - Sodium phosphate buffer 0.2M (Na2HPO4/NaH2PO4) vi dy pH t 5.0-8.0 - Thuc th ng: Hn hp A1:A2 = 4:1
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- Thuc th Arsenomolybdate : hn hp B3:B4 = 1:2. - A1: ha tan 15 g K-Na-tartrate4 H2O (Mui Rochelle ) v 30 g Na2CO3 (khan) vi khong 300 ml H2O. thm 20 g NaHCO3. ha tan 180 g Na2SO4(khan) vi khong 500 ml nc nng v ung si loi kh. Trn 2 dung dch trn sau khi lm ngui v hn hp c thm nc n 1 lt. - A2: 5 g CuSO4 5 H2O v 45 g Na2SO4 khan /250 ml H2O. - B1: 26.5 g (NH4)6Mo7O24 4 H2O /450 ml H2O. Va khuy va thm 21 ml H2SO4 m c. - B2: 3 g Na2HAsO4 7 H2O /25 ml H2O. - B3: Ha trn B1 v B2 vi nhau, trong t 37oC trong thi gian t 24 n 48 gi v bo qun trong ti - B4: 1.5 N H2SO4 (41 ml H2SO4 m c/lt). - 100 mM dung dch m Sodium Acetate pH 5.0 - 1% (w/v) tinh bt (tinh bt c ha tan trong nc, un nng v khuy u cho n khi h ha hon ton (dung dch trong, khng lng cn)) - Dung dch chun Glucose 0-1 mM trong 50 mM dung dch m Sodium Acetate pH 5.0. - Dung dch Acrylamide 30% - Dung dch m : - 1,5 M Tris-HCl, pH=8,8 - 0,6 M Tris-HCl, pH=6,8 - Amonium persulphat (APS) 10%. - SDS 10% - Dung dch Tetramethylethylenediamine (TEMED) - 1 lt dung dch chy in di c cha: - 6g Tris - 28,8g Glycerin - 1g SDS - 5ml dung dch chun b mu c cha: - 0,6M Tris-HCl, pH=6.8 5ml - SDS 10% 4ml - Glycerol 98% 2ml - -mercaptoethanol 1ml - H2O 8ml - Bromophenol blue 0.5% - 100ml dung dch nhum gel: 0,1 g comassie R-250 50 ml methanol
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40 ml nc ct 10 ml acid acetic 100% - 1 lt dung dch ra gel: - 100 ml methanol - 70 ml acid acetic 100%

2/ Phng php: a/ Phng php sc k: Ly trch enzyme:

-

g thc ny mm c nghin trong ci s vi ml dd m Tris-HCl ..M, pH .. Hn hp sau khi nghin mn c khuy t trong . pht, tip tc cho qua vi lc. Sau , ly tm ..v/pht trong . pht. Ghi li th tch, tr li 1ml o hm lng protein v hot tnh. Ly . ml chy sc k trao i ion.

Sc k trao i ion
-

Chun b gel . : Cho ..g gel vo cc thu tinh, ra gel bng dung dch m Tris-HCl M, pH .. Chun b ct .: Nhi gel vo ct thy tinh. Cn bng ct vi dung dch m Tris-HCl ..M, pH ... Tc dng chy .ml/pht Load mu: Cho .ml dung dch protein qua ct vi tc .ml/pht. Ra ct bng dung dch m Tris-HCl .M, pH ., theo di qu trnh ra ct qua sc k , cho n khi phn on protein khng bm trn ct c y ra ht khi ct. Tc dng chy l 1ml/pht. Thu protein khng bm. Ra gii enzyme: Ra gii bng gradient nng mui t .. - ..M NaCl (pha trong dung dch m Tris-HCl .M, pH ... Tc dng chy l .ml/pht Thu cc phn on protein nhn c, o .., o ., . trn gel SDS-PAGE. Chun b dung dch Bradford: . Chun b dung dch protein chun: Xy dng ng chun: nh lng protein c trong mu o Pha long cc mu theo bng sau:

b/ nh lng protein bng phng php Bradford

-

ng nghim

2 1 Protein Nc th ct (10ln)

3 Protein khng bm 1 (5 ln)

4 Protein khng bm 2 (5 ln)

5 Protein bm 1 (10 ln)

6 Protein bm 2 (10 ln)

Mu protein (l) Nc ct (l) o Th nghim lp li 2 ln. o Ly 100 l dung dch t cc ng nghim trn cho vo mt dy ng nghim c nh s tng t. o Cho vo mi ng 2ml thuc th. o Lc u ng bng my vortex. Trnh c bt. Sau 10 pht tin hnh o OD.

o Ghi nhn kt qu, ly gi tr trung bnh ca 2 ng

o Da vo phng trnh ng chun ta suy ra c hm lng protein

trong dung dch o. Ghi nhn kt qu. c/ Xc nh hot tnh enzyme Amylase - Dng ng chun glucose
o Pha dung dch ng chun: o Dng ng chun: o Ghi nhn kt qu - V biu ng chun.

Xc nh hm lng ng kh trong sn phm thy phn Tnh ton kt qu: Tnh ton hot tnh ring ca enzyme (U/mg).

d/ Phn tch protein bng phng php in di trn gel polyacrylamide (SDSPAGE) - . Chun b hp in di: -

Chun b gel phn tch polyarcrylamide 10 % Chun b gel tp trung

Chun b mu protein o Chun b cc mu protein theo bng sau:

ng Eppendorf

1 2 Protein Protein th bm 1

3 Protein bm 1 nh

4 Protein bm 2

5 Protein bm 2 nh

4 Protein khng bm 1 nh

5 Protein khng bm 2 nh

Mu protein (l) Buffer (l) -

a mu vo cc ging Tin hnh qu trnh in di: Sy gel. Chp hnh Gel v phn tch kt qu t c.

IV/ KT QU V THO LUN

1/ Phng php sc k
* Gi tr o OD ca cc mu Bng 1: Kt qu o OD cc phn on thu c trong qu trnh sc k
ng 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 Gi tr OD ng 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 Gi tr OD ng 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 Gi tr OD ng 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 Gi tr OD

-Da vo bng s liu ta v c biu nh sau:

Nng mui NaCl (M)

Nhn xt: ..

2. nh lng Protein bng phng php BradFord:

a./ Dng ng chun: Bng 2: Kt qu o OD 595nm ca cc mu protein chun

ng nghim Ln 1 Ln 2 Ln 3 Trung bnh

-Biu ng chun: th hin mi tng quan tuyn tnh gia nng protein (g/ml) v hp th (OD) bc sng 595nm

b./nh lng protein: Bng 3: Kt qu o OD 595nm ca cc mu protein c pha long Biu ng chun nng protein

Protein Th (10 ln)

Protein Khng bm 1 (5 ln)

Protein Khng bm 2 (5 ln)

Protein Bm 1 (10 ln)

Protein Bm 2 (10 ln)

Ln 1 Ln 2 Trung bnh -Da vo biu ng chun, ta c nng cc mu protein nh sau: Bng 4: Kt qu nng cc mu protein Th Th tch V (ml) Nng protein (g/ml) Protein tng (mg)

Khng bm 1

Khng bm 2

Bm 1

Bm 2

Nhn xt:

..

3/ Xc nh hot tnh ca enzyme amylase

a./ Dng ng chun: Bng 5: Kt qu o OD 520nm ca cc mu glucose chun ng 1 Ln 1 Ln 2 Trung bnh Nng ng glucose (mM) -Biu ng chun: th hin mi tng quan tuyn tnh gia nng glucose (mM) v hp th (OD) bc sng 520nm ng 2 ng 3 ng 4 ng 5 ng 6

Biu ng chun nng glucose

b/ Hot tnh enzyme: Bng 6: S liu o OD, nng ng kh v hot tnh ca cc mu. Kt qu OD 520nm Nng Hot Mu Hot tnh i Trung ng kh tnh protein tng U Ln 1 Ln 2 (mM) (U/ml) chng bnh Th Khng bm 1 Khng bm 2 Bm 1 Bm 2

Nhn xt:

4/ Phn tch protein bng phng php in di trn gel polyacrylamide (SDS-PAGE)
*Dng ng chun: Bng 7: Trng lng phn t v khong cch ca thang protein chun Band 1 Band 2 Band 3 Band 4 Band 5 Band 6 Band 7 Trng lng phn t M 116 66.2 45 35 25 18.4 14.4 (kDa) 2.06 1.82 1.65 1.54 1.39 1.26 1.16 Log M Khong cch 1.4 2.6 4 5.5 7.8 10.05 11.4 R (cm) 11.6 R0 (cm) Rf=R/R0 0.121 0.224 0.345 0.474 0.672 0.866 0.983

*Biu ng chun: th hin mi tng quan gia log ca trng lng phn t protein v t l khong cch gia cc band.

Biu ng chun ca log trng lng phn t vi Rf -Xc nh enzyme Amylase: Da vo ng chun ta c c kt qu cc band ca protein amylase nh hnh v.

Hnh gel in di

Nhn xt: -Theo l thuyt trng lng phn t ca enzyme -amylase l 54kDa v theo ng chun ta thy band trong hnh elip c trng lng l khong 53kDa. Ta c th suy ra y l band enzyme -amylase cn tm. - Ct protein th c nhiu band do cha nhiu tp cht. -Ct protein bm 1 v bm 1 nh xut hin band ca enzyme r hn v trong mu c cha lng ln enzyme -amylase. -Ct protein khng bm 1 v 2 khng xut hin band ca enzyme -amylase mc d chng c hot tnh l do hm lng enzyme -amylase trong khng bm rt thp. -Ct bm 2 khng xut hin band ca enzyme -amylase tuy vn c hot tnh, do hm lng protein trong mu thp nn khng thy band -amylase. Bng 8: Kt qu tng hp qu trnh tinh sch enzyme -amylase Tho lun: Kin ngh:

Mu Th Khng bm 1 Khng bm 2 Bm 1 Bm 2

Hm Protein Th tch lng tng V (ml) Protein (mg) (mg/ml)

Hot tnh (U/ml)

Hot tnh tng ( U)

Hot tnh c hiu (U/mg)

Hiu sut thu hi (%)

tinh sch

TI LIU THAM KHO