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1、沉淀蛋白质旳常用措施(TCA、乙醇、丙酮沉淀蛋白操作环节)TCA-DOCFor peciptaion overyl pein oncnratin 1) To e volumeof roioluti,add1/100vol. of 2% DO (Na dexycholate, detge). 2)Vorex an letsit for 30mn at o) Add 1/0 of richlroacec acd (C) 100%votxand et t ON a 4oC (preparton of 0 TCA： 44ml H2O/kg TA. Mantai in akbotleat C.Be ce
2、ul, ue gle!). 4) Spn 1mn 4C in crofug at aiuspe (1500）. Crefl dscarge uprnatant andretainte pellet: drytube b ersionn tiue paper (peet may be ifult oe). OPION: as plle tw toe volume of old aceton （atone ep at0oC). Vorte ad repelt amles min a full se bete ashe. 5) ry sampes undr vaccum(peedvac) or dr
3、y air. Fr PAGE-, esuspn samlesna minimal vlumeofampe bufer.（The presne o someTA anivayllo colou as a ceuenc ofhe acidifitio f the sme buer ；tirate wih 1N NaOH r MTrHC p8.5 tobtainthe noml blu sampe bfer colour.) NomlTC TeliminateTA solule neerncs ad otein oncentraton1) T sample of pein otion add Tri
4、ooaci ad (CA） 00% t get3% ial ocentraon. Mix an kep min20oC nd then 15mi 4oC;or longer ime at oC withot the2oC stp fr loer potein ccation. Sggestio: lee O iheptein concentrationis vey lo.(preprain f10% TCA: 54mlH2O/g CA intai dark botteat 4o.Becarful, e gloes!). 2)Spi 15m 4o in mifuge a xiuspeed (15
5、00g）.arefl dischge ueraan and retain he pellet: dry tube by nverion on tssue papr (lletmabe difiult o se) 3) or PAGE-SDS, rsuspen samples in aiimal vol of smple buffer(Te preenc o some TCA can ie a yellow colou as a osequece of theacidifatiof teampe bue ; tirte wt 1NaH o M TrisHC pH8. tobtin th rm b
6、ue samle bffer colu) ActonPrcipitton o eliminate aetoeolbleinterferecs an proten coentation1) Ad 1 volume f proei soluio 4 volumes ofold aceon. Mx an keep t east0min2o. (ugsio: leave O if the proen eratinisvey low). 2） Spi 15min 4C in mrofuge at axmum speed(150g). Carefully dishae supernatant n reai
7、nthe pelle: dy tube y iverion on tisue papr （elet aybdiffictosee).3) Dy aps un vccum (spevc) dry toelimie any acetone esidue(slltubes). o PAGD, supend saples n a minimal olume of smpl uffrhol Preipitation Useul method to onentrate proteis and remol ofGuanidneHydrolride beor AESDS1） Add to1 volme ofp
8、rotein slution 9voumesof cold Ethanol 10. ixand kee t last10min.at 0oC (Seston: leaeON). ) Sp 15min 4 in crcenriuge at axiumspe（00） Carefuy iscrge supnatantnd retan the peet: y ube b inver onissueapr（pelltmy fficultt ee).)sh peet with0% coldethanl （keep a20oC). rtex and reeletsaples 5mtfl speed.4）ry
9、 splesndervacm (spevc) dry air to eliminate any ethanolsid (sml es). r PAESDS, eupend amples i miml volume samplbufr. A-C/Acetone Uful methodo netra proten nd emov ceone ndTA souble interferen1. o one vlume f prtesolun dd 2% Na deoxychol (C) to0.02 final （for10 l mple, ad 1 2%DOC）. 2. ixn ketroom ep
10、eature for tast 15in.3.10tricoroic aid(TCA) to et 10% fal concntation (preparatio of 10% TCA: 454l H2O/g TCA Mainain i dbottlea 4oC.Be refu,uegls！!）. . Mx andeep at r mperrefot leas ur. . Spin t 4C for 1 min, reove supernaantand rain he pellet yte yinvrsio n isuepaper 6 Ad 200 l o ic colcetone to TC
11、 plle. . Mixnd eep on ceortleas 1 n. 8. Sp t 4for 10 in microcentrife maximum sped. . Remove uprnaat asbefo (), dry ai peleto lminteanacetoeresidu （smelltbs）.r PAGE-SD, ressped sples i a minmal vlumeof amle bffe. 10(heprence of some TC n givea yellow corasa consequenceof th aifictiono th samplbuffr;
12、 tirate wih1NaOH o MrisHC pH8 t obtith nmlblue smpleffer olr.） Acidife Actoe/MthanolUseful method to reme aetone and meanol soluble inerferences lie SDS before IEF ) Prpe acdified actone： 10ml aetone +0l Cl (1M final concentrton） 2) repare precipttion reagnt： ix qua vlume o aidiied acetone and ethanol andeep a -2oC. ) To n volume ofpotei solution d 4volumof coreciiion reagent. Min ep ON a 20oC ）Spin 1mi 4C inmicrfug t maxiumsped (1
