Spectrophotometric xim colourectometric protein ntau
Proteins yog feem ntau yog sib xyaw ntawm cov nqaijrog. Cov kev kuaj xim Colorimetric yog ua raws cov protein cov khoom noj: Cov amino acids (xws li, tyrosine, serine) nrog cov neeg chromogenic ntxiv los yog zas xim los tsim cov khoom xim. Qhov concentration ntawm cov khoom siv xim yog ncaj qha ntsig txog cov amino acids uas muaj protein ntau, ua li no xav txog cov protein concentration.
Colorimetric txoj kev
Muaj ntau txoj kev xws li BCA, Bradford, thiab Lowry.
Lowry txoj kev: Raws li qhov tshwm sim Biuret ntxov thiab zoo tuaj. Cov protein tshuam nrog Cu2 los ua cov kab xiav. Txawm li cas los, Lowry txoj kev yog ntau dua li Biuret. Qhov tsis zoo yog qhov xav tau los ntxiv ntau ntau yam reagents; cov tshuaj tiv thaiv no ntev heev; yog raug rau cov tshuaj tsis muaj protein; proteins uas muaj EDTA, Triton x-100, thiab ammonia sulfate tsis haum rau hom no.
Bicinchoninine acid assay: Qhov no yog qhov tshiab, ntau qhov kev xav tshuaj protein ntau. Cov protein yuav tsum tau soj ntsuam xyuas nrog Cu2 nyob rau hauv ib qho kua alkaline los tsim Cu, uas ua ib qho chelate nrog BCA los ua ib lub paj yeeb nrog lub nqus dej ntawm 562 nm. Txoj kab kev sib raug zoo ntawm qhov concentration ntawm no compound thiab protein yog qhov muaj zog, thiab cov compound tsim tom qab cov tshuaj tiv thaiv yog khov heev. Txheeb ze rau Lowry txoj kev, qhov kev lag luam yog qhov yooj yim thiab qhov muaj siab heev. Txawm li ntawd los, zoo li Lowry txoj kev, nws yog susceptible rau cuam tshuam nrog proteins thiab detergents.
Txoj kev cai Bradford: Txoj ntsiab cai ntawm qhov txujci no yog tias cov khoom muaj protein ntau nrog Coomassie Brilliant Blue los tsim cov xim dawb uas absorbs ntawm 595 nm. Qhov loj tshaj plaws feature yog nws rhiab heev, uas yog 2 lub sij hawm uas ntawm Lowry thiab BCA. Nws yog yooj yim thiab sai dua. Nws tsuas yog yuav tsum muaj ib hom tshuaj tiv thaiv. Lub compound yuav ruaj khov rau 1 xuab moos los pab txhawb qhov kev tshwm sim; Interfering nrog Lowry, BCA reacts nrog txo cov neeg ua hauj lwm (xws li DTT, mercaptoethanol) sib xws. Txawm li cas los, nws yog tseem sensitive rau detergents. Lub ntsiab tseem ceeb yog tias cov qauv sib txawv tuaj yeem ua rau muaj kev sib txawv loj hauv cov qhab-nees ntawm tib tus qauv thiab tsis pom cov qhab nia.
Qee cov neeg tshawb nrhiav uas raug cov kev ntsuas cov xim thaij thawj zaug yuav tsis meej pem los ntawm kev ntsuam xyuas ntau yam xim ntawm cov xim Colorimetric. Yam dab tsi uas koj ntseeg hais tias yuav tsum tsis meej pem? Vim tias cov pab pawg tau ua ntau yam thiab cov chromogenic pawg tsis zoo li qub, ob peb txoj kev siv tib lub sij hawm los ua cov qauv ntau ntawm cov qauv tib yam inconsistent. Piv txwv, Keller li al. ntsuam xyuas cov protein nyob hauv tib neeg cov mis thiab pom hais tias cov kev ntsuas tau ntsuas los ntawm Lowry thiab BCA tau siab dua li ntawm Bradford. Qhov txawv yog qhov tseem ceeb. Txawm hais tias tib qho qauv txiav txim siab, tus qauv coj los xaiv los ntawm tib qho xim Colorimetric yog inconsistent, thiab cov concentration tom qab qhov kev kuaj kuj yog tsis sib xws. Piv txwv, siv Lowry ntsuam xyuas cov protein nyob hauv cell homogenate, siv BSA raws li tus qauv, nrog cov concentration ntawm 1.34 mg / ml, thiab globulin raws li tus qauv nrog concentration ntawm 2.64 mg / ml. Yog li, ua ntej xaiv ib txoj kev colorimetric, nws yog qhov zoo rau xa cov tshuaj lom neeg muaj pes tsawg leeg ntawm cov qauv coj mus kuaj, thiab nrhiav tau tus qauv protein zoo sib xws li tus txheej txheem. Tsis tas li ntawd, txoj kev uas siv cov xim thaij rau ntau yam ua rau cov nqaij tawv ntau npaum li cas yuav ua rau cov teebmeem ntawm qhov hnoos qis tsis dhau, uas ua rau muaj qhov sib txawv ntawm qhov ntsuas cov ntsuas thiab qhov tseeb cov concentration. Qhov teeb meem tseem ceeb yog qhov xim ntawm qhov tseem ceeb ntawm tus 1011 spectrophotometer, lub cuvette, muaj ib feem ib nrab ntawm lub neej, yog li txhua txoj kev xaiv xim teev npe rau lub sijhawm xeem. Tag nrho cov qauv (nrog rau cov qauv kuaj) yuav tsum kuaj hauv lub sijhawm no. Yog hais tias lub sij hawm ntev dhau lawm, tus nqi absorbance tau me dua thiab cov nqi hloov dua siab tshiab tau hloov. Tsis tas li ntawd, qhov ntsuas kub, qhov pH nqi ntawm kev daws, thiab lwm yam yog txhua yam tseem ceeb uas cuam tshuam rau kev sim. Tsis tas li ntawd, nws yog ib qho tseem ceeb heev uas yuav tau siv cov yas xim. Txhob siv cov cuvettes ua los ntawm quartz los yog iav vim tias qhov xim tom qab qhov tshuaj tiv thaiv yuav ua rau cov xim quartz los yog iav kom muaj xim, ua rau tsis muaj qhov hnoos qeev.
Lub tuam txhab tshwj xeeb hauv kev muag khoom ntawm spectrophotometers , txais tos cov neeg siv khoom uas muaj qhov xav tau los tham nrog thiab yuav khoom, peb yuav muab koj nrog sincere cov kev pab cuam thiab cov khoom zoo, nrog rau cov nqi qis dua.
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