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1. Nguyn tc Phng php ny da trn c s phc cht ng protein kh hn hp photphomolipden photphovonphramat (thuc th Folin ciocalteu) to phc cht mu xanh da tri c hp th cc i bc song 660nm. Cng mu ca hn hp phn ng t l thun vi nng protein trong mt phm vi nht nh. Da vo mc hp th quang hc ca protein chun, ta c th xc nh c hm lng protein trong mu nghin cu.
2. Ho cht cn dng - Dung dch A: 4g NaOH (0,1M) v 20g Na2CO3 (2%) pha trong 1000ml nc ct. - Dung dch B: 0,5g CuSO4.5H2O (0,5%) pha trong dung dch Natri Xitrat (1%) hoc trong dung dch Natri, Kali Tactrat 1%. - Dung dch C: Hn hp ca hai dung dch A v B theo t l 49:1 (pha trc khi dng). - Thuc th folin, trc khi dng pha long hai ln sao cho axit bng 1N. - Albumin huyt thanh b 1mg/ml.
- Mu th nghim: ly chnh xc 0,5ml dch cha protein vi hm lng thch hp cho vo ng nghim, thm vo 2ml dung dch C, lc u yn trong 10 pht. Sau thm vo hn hp trong ng nghim 0,25ml folin pha long 2 ln, lc u v yn trong 30 pht, mu vng ca hn hp chuyn sang mu xanh da tri v t n cng mu cc i. em so mu ca hn hp trn my so mu quang in bc sng 660nm. Xc nh c tr s mt quang hc ca dung dch nghin cu. o trn my 3 ln lp li v ly tr s trung bnh. - Mu i chng: 0,5ml m cho vo ng nghim, thm vo 2ml dung dch C v bc tip theo c tin hnh nh mu th nghim.
4. Xy dng ng th chun Hm lng protein tan trong dung dch c xc nh theo phng php Lowry, dng albumin huyt thanh b (BSA) lm cht chun. Cn 0,01g (10mg albumin huyt thanh b) ho tan trong 10ml nc, ta c dung dch gc c nng l 1mg/ml. Sau pha long dung dch gc bng nc ct vi cc nng 20, 40, 60, 80, 100, 120 g/ml tin hnh xy dng th chun. - Mu th nghim: ly chnh xc 0,5ml dung dch BSA nng pha long nh trn cho vo ng nghim, thm vo mi ng 2ml dung dch C nhit phng 10 pht, sau cho 0,25ml thuc th folin (1N) em so mu vi bc sng 660nm. - Mu i chng: ly 0,5 ml nc ct cho vo ng nghim, thm vo 2ml dung dch C v cc bc tip theo c tin hnh nh mu th nghim. Qua 3 ln lp li th nghim c th xy dng ng hi quy. Kt qu c x l theo phng php thng k thng thng.
y = 0,55066844x + 0,01791961
OD 660 nm
mg
Phng php ny da vo s thay i mu sc xy ra khi Coomassie Brilliant Blue G-250 lin kt vi protein trong dung dch axit, dng proton ha ca thuc nhum Coomassie Blue c mu da cam . Thuc nhum lin kt cht ch vi cc protein, tng tc vi c nhm k nc v cc nhm mang in tch dng trn phn t protein.Trong mi trng ca cc gc mang in tch dng, s proton ha khng xy ra v c mu xanh xut hin.
2. Vt liu v dng c: - Cc mu protein. - Dung dch mu( thuc nhum) + Coomassie Brilliant Blue G-250 (CBB G-250)100mg + Ethanol (EtOH) (absolute) 95%50ml + H3PO4 85% + H2O - Pipet man (Hng Gilson Php). - My o quang ph Shimadzu UV 200s (Japan). - ng nghim
3. Cch tin hnh 3.1. Xy dng ng cong chun biu din nng protein chun (BSA): * Quy trnh phn tch:
Dung dch protein chun BSA (Bovine Serum Albumin) 10-100g protein/ml dung dchNaCl 0,15M
ng th nghim: 0,1ml dung dch protein chun ln lt cc nng t 10-100g, thm 5ml dung dch thuc nhum, trn u, phn ng xy ra nhit phng.
ng i chng: 0,1ml dung dch NaCl 0,15M, thm 5ml dung dch thuc nhum, trn u, phn ng xy ra nhit phng.
Dung dch protein chun BSA (Bovine Serum Albumin) 1,0-10,0 g protein/ml dung dchNaCl 0,15M ng th nghim: 0,1ml dung dch protein chun ln lt cc nng t 1,0-10,0g, thm 1ml dung dch thuc nhum, trn u, phn ng xy ra nhit phng.
ng i chng: 0,1ml dung dch NaCl 0,15M, thm 1ml dung dch thuc nhum, trn u, phn ng xy ra nhit phng.
c quang ph bc sng 595nm sau 2 pht n 1 gi (cuvet 3cm). => V th biu din s bin thin mt quang (OD) , theo nng protein chun BSA tng ng vi gi tr OD 595nm. (2 th tng ng vi 2 dung dch protein chun)
ng cong tiu chun ny c s dng xc nh hm lng protein trong cc mu cha bit, cng c xy dng tng t theo phng php Lowry.( hnh 7.2 v 7.3 trang 88 sch thc hnh ha sinh hc-Nguyn Vn Mi) 3.2. nh lng protein camu:
Ht 0,1ml mu protein cn phn tch vo ng nghim Cho thm t 1-5ml thuc nhum, lc u, ch khong 10 pht o quang ph bc sng 595nm
X=[C].a
Trong : + X: s g protein c trong mu + [C]: nng protein theo th (g/ml). + a: pha long dung dch. Mun xc nh hm lng protein kh ta tnh:
P=X.V/m
Trong : + X: s g protein c trong mu + V: thtch dung dch trch ly; ml + m: khi lng mu; g
Lu : - Thuc nhum phn ng vi protein to dung dch c mu xanh. - Cng mu t l thun vi nng protein c trong dung dch. - y l th nghim vi hp cht mu, nn ng nghim v cuvet phi c loi b cht mu bng axeton, sau ra hoc ngm trong HCl 0,1M. nh gi: Phng php ny c nhy cao, cho php pht hin 5-200g protein/ml, d thc hin v tit kim thi gian.
Phng php ny l cc axit amin thm nh tryptophan , tyrosine , phenylalanine trong chui polypepetid c ph hp th bc sng cc i bc sng 282nm. hp th tia cc tm thay i ty loi protein , nhng h s tt o c (ngha l hp th ca dd protein 1% vi ng truyn qua 1cm),cho mi protein cho php tnh nng ca protein tinh sch , s hp th tia cc tm : protein hp th tia cc tm bc sng 240-280nm , axit
amin nhn thm :tryptophan , tyrosine , phenylalanine c ph hp th 280nm . ng thi chng cng hp th bc sng thp hn 215-230nm hp th ny ph hp cho peptid khng c tryptophan , tyrosine. Nu protein tinh sch th nng tuyt i ca n tnh theo gi tr o c . Nu protein khng tinh sch th nng ca protein tng s c tnh tng i t hp th . Phng php ny khng dung c cho cc dd c nng protein thp <0.1mg/ml hoc khi c mt nhiu cht khc m hp th cng 1 vng cc tm hoc khi protein trong dch huyn ph ch khng phi trong dd . 2. Nguyn liu v thit b : DD protein o m ha tan protein My quang ph UV c cuvet thch anh ng sang truyn qua 1cm 3. Tin hnh :
Li tm mu loi b cc phn t hoc phc hp khc c trong th
huyn ph t bc sng 280nm my quang ph v iu chnh hp th v khng vi cuvet cha m c hp th ca mu hoc trong cng mt cuvet hoc cuvet khc cng cp . Nu gi tr thu c > 2.0 th pha long mu (1/5 hoc 1/10) hoc ng sang truyn ngn hn (2nm) cho n khi s c nm trong khong 0.1-0.5 Lp li bc 2 v 3 260nm
Nu t s cao hn cho bit protein khng sch , c ln tp cht khc c bit vi axit nucleic . Nng mu = ( hp th 280nm/h s tt 280nm)*10mg/l Vi mt hn hp cc protein hoc vi bt c mt loi protein no m khng bit h s tt th tnh nh sau :
nh lng H2SO4 cn li bng NaOH , qua tnh c lng nito c trong mu H2SO4 d + NaOH = Na2SO4 + H2O
Sau khi thm xc tc un nh hn hp , trnh si tro v ch un mnh khi hn hp hon ton chuyn sang dch lng . Trong qu trnh un thnh thong lc nh , trng kho lo sao cho khng cn 1 vt en no ca mu nguyn liu cha b phn hy cn st li trn thnh bnh . un cho ti khi dd trong bnh hon ton trng. Chuyn ton b dd v c ha xong vo bnh nh mc 100ml , thm nc ct cho n vch nh mc . Ch phi lm ngui v lc u. 3.2. Ct m :( tm hiu th nghim ha sinh) 3.2.1. Ra sch bnh Parnas Wagner. 3.2.2. Ct m Cho 10ml dd H2SO4 0,1N + 3 git Phenolphthalein vo erlen E. Ht 10ml dd v c ha cho vo phu C. un si A. Cho 10ml NaOH d vo C. nh phn H2SO4 0,1N d = dd chun NaOH 0,1N . 4. Tnh ton 4.1 Tnh h s hiu chun F ca dd NaOH - Ly 10ml H2SO4 0.1N chun cho vo erlen , nh phn bng NaOH 0.1N - Tnh nng thc t ca dd NaOH em nh phn - F l t s gia nng thc t v nng l thuyt ca NaOH 4.2 Tnh kt qu :
X = (a-b.F).0.0014.100.1000/10.V
Trong : X : hm lng nito tnh bng g/l a : s ml H2SO4 em hp th NH3 b : s ml NaOH 0.1N tiu tn khi chun H2SO4 tha V :s ml mu em v c ha 0.0014 : lng gam nito ng vi 1ml H2SO4 F : h s hiu chnh nng dd NaOH 0.1N Tnh hm lng protein th thng qua hm lng nito tng Nito trong vt liu sinh hc ch yu l nito protein ngoi ra cn c 1 lng nh nito trong cc thnh phn khc gi l nito phi protein . Nito tng = Nito protein + Nito phi protein Hm lng protein trong cc vt liu sinh hc c tnh bng cch nhn hm lng nito protein vi 1 h s chuyn i xc nh do hm lng nito c t l n nh t 15 18% protein , i a s protein l 16% Trong cc nguyn vt liu sinh hc do hm lng nito phi protein nh v vic tch ring rt phc tp nn theo quy c ngi ta tnh hm lng protein theo nito tng v gi l protein th hay protein tng . Cng thc tnh hm lng phn trm protein th c trong i a s cc nguyn liu l :
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