1.

Phng php xc nh hm lng protein theo Bradford

Trong cc phng php xc nh hm lng protein th y l phng php c
nhy cao, c th xc nh ti 1g, ho cht n gin t tn thi gian. Mt u im ln ca
phng php ny l t b cn tr bi cc ho cht s dng trong nghin cu protein, nht
l amoniumsufate.
Nguyn tc:

Phng php ny da trn s thay i bc sng hp thu cc i ca thuc nhum

Coomassie Brillant Blue khi to phc hp vi protein. Trong dung dch mang tnh acid
khi cha kt ni vi protein th thuc nhum dng mu c bc sng thp thu cc
i l 465 nm v khi kt hp vi protein th thuc nhum chuyn sang dng mu xanh
dng v hp thu cc i mc cc i bc sng 595 nm. hp thu bc sng
596 nm c lin h mt cch trc tip vi nng protein.
xc nh protein trong mu, u tin ta xy dng mt ng chun vi dung
dch protein chun bit c nng . Sau khi cho dung dch protein vo thuc nhum
mu, mu s xut hin sau 2 pht v bn ti 1gi. Tin hnh o dung dch bng my
quang ph k (Spectrophotometer) ta c OD x, hp th s t l vi lng protein
trong mu. Thc hin mt i chng vi HCl (OD o). Ly gi tr OD = ODX ODO.
Lng protein trong mu dung dch o c xc nh bng cch da vo ng chun t
gi tr OD trc tung, t suy ra gi tr nng protein tng ng trn trc honh.
Ha cht

Dung dch albumine 0.1%.

Nc ct
Dung dch thuc th Bradford: thnh phn thuc th trong 100 ml nh sau
o

Coomassie Brilliant Blue: 0.001g.

Ethanol tuyt i:

4.7 g.

Phosphoric acid:

85%.

Phm mu Coomassie Brilliant Blue c ho tan trong ethnol trong mt chai

ng mu ti c np, b sung phosphoric acid v chnh ti 100 ml bng nc ct. Lc
u v gi lnh di 4oC.

Dng ng chun albumine

Pha long dung dch albumine thnh cc nng khc nhau: 0, 10, 20, 30, 40, 50,
60, 70, 80, 90, 100 g/ml.
Bng 3.1. Dng ng chun albumine
ng nghim

10

20

30

40

50

60

70

80

90

10

20

30

40

50

60

70

80

90

1000

990

980

970

960

950

940

930

920

910

chng

Nng (g/ml)
Dung dch
albumin chun
(l)
Nc ct (l)
Thuc th
Bradford (ml)

ng s 0 tng ng vi ng i chng cha nc ct. Tt c cc ng sau khi pha

ng nng , khong 20 pht.
Tin hnh o mt quang ca dung dch bc sng 595 nm.
Dng ng chun.
nh lng protein trong mu th nghim
Ly 1ml mu th nghim cho vo ng nghim, thm vo 2 ml thuc th Bradford. em
o mt quang bc sng 595 nm.
T suy ra hm lng protein c trong mu th nghim.
Mu c pha long sao cho mt quang o c trong khong ng chun.
Kt qu tnh ton:

T phng trnh ng chun v mt quang ca mu kho st ta suy ra hm lng

protein ca mu l X (g/ml) c trong m (g) nguyn liu.
Vy lng protein c trong 1 gam nguyn liu l:
Hm lng protein (mg/g) =

X
1000. m

Hnh 3.2. ng chun albumine

2. Xc nh hot tnh enzyme protease bng phng php Amano

Nguyn tc:

Dng protein casein lm c cht xc tc nh hot tnh phn gii protein ca

enzyme protease trn c s nh lng sn phm to thnh trong phn ng bng phn
ng mu vi thuc th Folin. Da vo th chun tnh lng Tyrosin tng ng vi
lng sn phm thu phn di tc dng ca enzyme.

Ho cht:

HCL 0.1 M

Ma2CO3 0.4 M

Dung dch Trichloracetic 0.4 M

Tyrosin tinh khit

NaOH 0.1 M

Thuc th Folin

H3PO4 1/30 M

m phosphate (pH 7) 0.1 M

Dng c v thit b

ng nghim, pipette, bnh nh mc, giy lc.

B n nhit

My o quang ph UV Vis

My o pH

Xy dng ng chun tyrosine

Bng 3.2. ng chun tyrosine
ng s

10

20

30

40

50

60

70

80

90

10

20

30

40

50

60

70

80

90

Dung dch HCl (l)

1000

990

980

970

960

950

940

930

920

910

Na2CO3 (ml)

Nng (g/ml)
Dung dch tyrosine
chun (l)

Thuc th Folin
(ml)

chng
0

Pha dung dch tyrosine cc nng khc nhau : 10, 20, 30, 40, 50, 60, 70, 80,
90, 100 gtyrosine / mlHCl nh bng 3.3.2. Thm 5 ml dung dch Na 2CO3 0,4M vo dung dch
tyrosine cc nng khc nhau trn v thm 1 ml thuc th Folin vo dung dch hn
hp. Sau khi trn u, n nh dung dch 37 0,50C trong 20 pht. o hp th

ca dung dch ny bc sng 660 nm. Ghi nhn kt qu As10, As20, As30, As40, As50, As60,
As70, As80, As90. As100
i vi ng i chng dng 1 ml acid HCl 0,1M thay cho tyrosine. o hp thu
ca dung dch ny bc sng 660 nm v ghi nhn kt qu ny l Aso.
Tr s mt quang ca cc ng t 1 n 9 sau khi tr i tr s ca ng i chng s
c gi tr OD1 n OD9. V th da vo bin thin ca OD theo nng
protein. th ny gi l ng chun tyrosine.
Xc nh hot tnh enzyme protease
Hot tnh enzyme c kho st nhit 370C v pH = 7.
Cho 1 ml dung dch c cht casein 1% vo ng nghim, 37 0,50C trong 10
15 pht.
Sau thi gian , cho 1 ml dch chit enzyme th vo v lc u. em hn hp ny
37 0,50C trong mt gi.
Cho vo 2 ml dung dch TCA 0,4M ngng phn ng enzyme.
n nh dung dch trong 25 pht, sau lc dung dch ny qua giy lc loi ta.
Cho 5 ml dung dch Na2CO3 vo 1ml dch lc.
Thm 1 ml thuc th Folin.
Trn u, yn 37 0,50C trong 20 pht.
Khi dung dch xut hin mu xanh, em o hp thu bc sng 660 nm (ghi
nhn kt qu ny l Am).
Mu i chng : ly 1 ml nc ct thay cho 1 ml dung dch enzyme v tin hnh
cc bc tng t nh mu th nghim vi cng iu kin. Ghi nhn kt qu hp th
l A0.
Hm lng tyrosine c gii phng do protease thy phn c tnh bng cch
ly Am A0 ri ly kt qu so vi ng chun tyrosine xc nh hot tnhprotease
theo tnh ton sau :

Mt n v hot tnh (VHT) enzyme protease c xc nh l lng enzyme

to ra lng amino acid tng ng vi 100 g tyrosine trong 1 ml dch lc di iu
kin th nghim. K hiu l: U
Hot tnh enzyme protease (VHT/ml) = (Am A0).F.1/100.n
(Am A0).F.1/100.n.V
m
F = [10/ As10 + 20/ As20 + 30/ As30 + 40/ As40 + 50/ As50 + 60/ As60

Hot tnh enzyme protease (VHT/g) =

+ 70/ As70 + 80/ As80 + 90/ As90+100/As100]/10

Am : hp thu ca mu
A0 : hp thu ca ng i chng
F : H s tng quan gia hm lng tyrosine v hp thu bc sng 660
nm trn ng chun.
n : H s pha long ca enzyme
1/100 : H s chuyn i
V : Th tch ca dung dch enzyme
m : khi lng ch phm enzyme th.
Tnh ton hot tnh ring (HTR) ca enzyme protease: t kt qu hm lng
protein (mg/ml) v hot tnh enzyme protease (U/ml) tnh hot tnh ring ca enzyme.
HTR (U/mg) =

S n v hot tnh
mg protein enzyme

ng chun tyrosine