肌原纤维蛋白的提取

肌原纤维蛋白提取1.3.1肌原纤维蛋白提取肌原纤维蛋白提取方法参考Liu等回的方法并略作修改，取猪背最长肌，剔除脂肪和结缔组织，切成1cm5小决.加入4倍休枳缓冲液(lOmmol/LNmPO」、0.lmo]/LNaCK2mmo]/LMgCL^111mmo]/LEGTA.pH7.0)，匀浆60s,200。Xg冷冻高心ISmim取沉淀重复上面步骤两次，得到粗提的肌原纤维蛋白口然后取此沉淀加入4倍体枳的0Imol/LNaCl^f液.匀浆60s.2000Xg冷7东离心L5min,重复此操作一遍，取沉淀加4倍体积的OJmol/LNaCl溶液,匀浆4层纱布过滤’取上清液,用0.Imol/LHC1调节pH值至6.0,2000Xg冷冻离心15min,沉淀即为提纯的肌原纤碓蛋白，4'C冷藏备用。以上操作均在4C进行.用双缩腿法以标准牛血清白蛋白为标准蛋白测定肌原纤维蛋白含量廿2、熊友林老师方法PreparationofMyofibrils.Myofibrilisolateswerepreparedina23Cwalk-incooler.Thawedmusclewasfinelychoppedwithaknife,mixedwith4vol(w/v)ofisolationbuffer,andblendedinaWaringblenderfor30sathighspeed.Theisolationbuffercontained0.1MNaCI;2mMMgCI?1mMEGTA,6.1mMsodiumphosphatedibasic(Na2HPO4),and3.9rnMsodiumphosphatemonobasic(NaH2PO4H2O)(pH7,0).Aftercentrifugationat2000gfor15min,thecrudemyofibrilpelletwaswashedtwomoretimeswith4volofthesameisolationbufferusingthesameblendingandcentrifugationconditionsindicatedabove.Themyofibrilpelletwasthenwashedthreemoretimeswith4volof0.1MNaCIunderthesameconditionsasaboveexceptthatinthelastwash,themyofibrilsuspensionwasfiilteredthroughfourlayersofcheeseclathtoremoveconnectivetissueanditspHwasadjustedto6.0(tocloselysimulatethepHconditioninprocessedmeats)with0.1NHCIpriortocentrifugation.Myofibrillarproteinisolate(MPI)waskeptinatightlycappedbottleandstoredonicebeforeuse(in18h).TheproteinconcentrationofthemyofibrilpelletwasmeasuredbytheBiuretmethod(23)usingbovineserumalbumin(SigmaChemicalCoSt.Louis.MO)asastandard.结合小白师姐文章：Gelationofsaltedmyobrillarproteinundermalondialdehyde-inducedoxidativestressRawmusclewasthawedat4andthenusedforthemyofi-mH^rproteinpreparationaccordingtotheme-thodofPark,Xiang.Mderton,andOoizumi(2006).ThepHofmyofibrillarproteinsuspensionat0.1MNaClinthelastwashwasadjustedto6.25beforezentrifugation.Thepalletwasfinallysuspendedin50mMsodiumphosphatebuffer(PB)(pH6.0)andtheproteinconcentrationwasieterminedbytheBiuretitiethodusingBSASSStandard.3、小白师姐风味吸附文章(参考)PreparationrfMyofibrillarProteins.Rawwasthawedat4°CandthenusietlforthemyofibrillarproteinpreparationiccordingtuthemethodofPark,Xion^Aided:on^andOoizumi22withslightrnodL£GatLU[is.ThepHoFmyo£brillarproteinsuspensionat0.1MNaClinthelastwashwasadjustedtu6.25beforecentrifugation.Thepelletw昭finallysuspendedin20mMsodiumphosphatebuffer(pH6.-0)containing0.5MNaCl,andtheproteinconeentrationwasdetennlnedbytheBiuretmethodusingBSAasstandard.lh.eproteincomponentofCantonesesausagewerefraction—atcdaccordingtothemethodofViscssanguanctal.[2004]withslightmadification.Samples(LQg)wereextractedwirhI(JOmlofphosphatebuffersalcti□□A(15.6mMN击HI'd.3.5mMKH^POj,pH7.5〕usinganUltri-Turraxh-omogenizer(BeijingJin.-gkeHiJ3.ru1InstrumenTCo.Ltd,Beijing,China)at8000rpmfor1minin.icebath.Thehomegen^rcwascenrrifugeddtSOflOgfor15minat4°CusingaCL-21Mcentrifuge(XiangYiCentrifugeInstrumentCo.Ltd,.Changsha.Chiru).Theextractionwasrepeatedtwice.Thesupernatants[wareT-solublenirragen)werecombined.Analiquot(50ml)ofrhesupernatanrwaskeptforsubsequentnitrogenri.crerrninarionrwhiletheresrwasmixedwithacold50%TCAtoafinalconcentrationofiCl^.Theresultingprecipitatewa5collectedbycenrrifugacianat5000gforISmin(thewater-solubleprorein).Thefilrrarewasthenon-proteinnitrogen[NPN}fraction.Thepelletwas^xrract^dwith100mlofphosphatebuffer5olurinnB(0.45MKCl15.6mtvtN迫15mMKhg.pH7」5)at8000rpmusinganUltra-Turraxhomogenizerfor1mininicebathandcentrifugedat500Qgfor15minit4u^ingaGL-21MCEntrifuge.Theextracrinn.wasrepeatedtwice.Thesuperna-ta.ni5werecombinedroobtainsalr-5olub]eprarein.ThepEl政at>tailiedwasextractedwith100ndcf0.1MNaOHwithcantinuousstirringfor8h.Ihc-mixrurrwasc^nrrifugrdandthe-superrurantwascolleeredasalka]i-solubleprotein.Thefinalrcsidu-ewasrhealkali-insolubleFraction.Thenitrogen匚口n怔ntofalltheproteinandnon-proreinfractionswasdeterminedbyKjNdati]method(LiueraL2A07XAlltheproteinfractionswereanalysedbySDS-polyacryIamidegelelectrophore^ifaccordingtothemethodofRore5etal.(200