沉淀蛋白质的常用方法

沉淀蛋白质的常用方法 沉淀蛋白质的常用方法 TCATCA 乙醇 丙酮沉淀蛋白操作步骤 乙醇 丙酮沉淀蛋白操作步骤 2010 08 18 15 19 TCA DOCTCA DOC ForFor precipitationprecipitation ofof veryvery lowlow proteinprotein concentrationconcentration 1 1 ToTo oneone volumevolume ofof proteinprotein solution solution addadd 1 1001 100 vol vol ofof 2 2 DOCDOC Na Na deoxydeoxy cholate cholate detergent detergent 2 2 VortexVortex andand letlet sitsit forfor 30min30min atat 4oC 4oC 3 3 AddAdd 1 101 10 ofof TrichloroaceticTrichloroacetic acidacid TCA TCA 100 100 vortexvortex andand letlet sitsit ONON atat 4oC4oC preparation preparation ofof 100 100 TCA TCA 454ml454ml H2O kgH2O kg TCA TCA MaintainMaintain inin darkdark bottlebottle atat 4oC Be4oC Be careful careful useuse gloves gloves 4 4 SpinSpin 15min15min 4oC4oC inin microfugemicrofuge atat maximummaximum speedspeed 15000g 15000g CarefullyCarefully dischargedischarge supernatantsupernatant andand retainretain thethe pellet pellet drydry tubetube byby inversioninversion onon tissuetissue paperpaper pellet pellet maymay bebe difficultdifficult toto see see OPTION OPTION WashWash pelletpellet twicetwice repelletrepellet samplessamples 5min5min atat fullfull speedspeed betweenbetween washes washes 5 5 DryDry samplessamples underunder vaccumvaccum speed speed vac vac oror drydry air air ForFor PAGE SDS PAGE SDS resuspendresuspend samplessamples inin a a minimalminimal volumevolume ofof samplesample buffer buffer The The presencepresence ofof somesome TCATCA cancan givegive a a yellowyellow colourcolour asas a a consequenceconsequence ofof thethe acidificationacidification ofof thethe samplesample bufferbuffer titratetitrate withwith 1N1N NaOHNaOH oror 1M1M TrisHClTrisHCl pH8 5pH8 5 toto obtainobtain thethe normalnormal blueblue samplesample bufferbuffer colour colour NormalNormal TCATCA ToTo eliminateeliminate TCATCA solublesoluble interferencesinterferences andand proteinprotein concentrationconcentration 1 1 ToTo a a samplesample ofof proteinprotein solutionsolution addadd TrichloroaceticTrichloroacetic acidacid TCA TCA 100 100 toto getget 13 13 finalfinal concentration concentration MixMix andand keepkeep 5min5min 20oC20oC andand thenthen 15min15min 4oC 4oC oror longerlonger timetime atat 4oC4oC withoutwithout thethe 20oC20oC stepstep forfor lowerlower proteinprotein concentration concentration Suggestion Suggestion leaveleave ONON ifif thethe proteinprotein concentrationconcentration isis veryvery low low preparation preparation ofof 100 100 TCA TCA 454ml454ml H2O kgH2O kg TCA TCA MaintainMaintain inin darkdark bottlebottle atat 4oC Be4oC Be careful careful useuse gloves gloves 2 2 SpinSpin 15min15min 4oC4oC inin microfugemicrofuge atat maximummaximum speedspeed 15000g 15000g CarefullyCarefully dischargedischarge supernatantsupernatant andand retainretain thethe pellet pellet drydry tubetube byby inversioninversion onon tissuetissue paperpaper pellet pellet maymay bebe difficultdifficult toto see see 3 3 ForFor PAGE SDS PAGE SDS resuspendresuspend samplessamples inin a a minimalminimal volumevolume ofof samplesample buffer buffer The The presencepresence ofof somesome TCATCA cancan givegive a a yellowyellow colourcolour asas a a consequenceconsequence ofof thethe acidificationacidification ofof thethe samplesample bufferbuffer titratetitrate withwith 1N1N NaOHNaOH oror 1M1M TrisHClTrisHCl pH8 5pH8 5 toto obtainobtain thethe normalnormal blueblue samplesample bufferbuffer colour colour AcetoneAcetone PrecipitationPrecipitation ToTo eliminateeliminate acetoneacetone solublesoluble interferencesinterferences andand proteinprotein concentrationconcentration 1 1 AddAdd 1 1 volumevolume ofof proteinprotein solutionsolution toto 4 4 volumesvolumes ofof coldcold acetone acetone MixMix andand keepkeep atat leastleast 20min20min 20oC 20oC Suggestion Suggestion leaveleave ONON ifif thethe proteinprotein concentrationconcentration isis veryvery low low 2 2 SpinSpin 15min15min 4oC4oC inin microfugemicrofuge atat maximummaximum speedspeed 15000g 15000g CarefullyCarefully dischargedischarge supernatantsupernatant andand retainretain thethe pellet pellet drydry tubetube byby inversioninversion onon tissuetissue paperpaper pellet pellet maymay bebe difficultdifficult toto see see 3 3 DryDry samplessamples underunder vaccumvaccum speed vac speed vac oror drydry airair toto eliminateeliminate anyany acetoneacetone residueresidue smell smell tubes tubes ForFor PAGE SDS PAGE SDS resuspendresuspend samplessamples inin a a minimalminimal volumevolume ofof samplesample buffer buffer EthanolEthanol PrecipitationPrecipitation UsefulUseful methodmethod toto concentrateconcentrate proteinsproteins andand removalremoval ofof GuanidineGuanidine HydrochlorideHydrochloride beforebefore PAGE SDSPAGE SDS 1 1 AddAdd toto 1 1 volumevolume ofof proteinprotein solutionsolution 9 9 volumesvolumes ofof coldcold EthanolEthanol 100 100 MixMix andand keepkeep atat leastleast 10min at10min at 20oC 20oC Suggestion Suggestion leaveleave ON ON 2 2 SpinSpin 15min15min 4oC4oC inin microcentrifugemicrocentrifuge atat maximummaximum speedspeed 15000g 15000g CarefullyCarefully dischargedischarge supernatantsupernatant andand retainretain thethe pellet pellet drydry tubetube byby inversioninversion onon tissuetissue paperpaper pellet pellet maymay bebe difficultdifficult toto see see 3 3 WashWash pelletpellet withwith 90 90 coldcold ethanolethanol keep keep atat 20oC 20oC VortexVortex andand repelletrepellet samplessamples 5min5min atat fullfull speed speed 4 4 DryDry samplessamples underunder vaccumvaccum speed speed vac vac oror drydry airair toto eliminateeliminate anyany ethanolethanol residueresidue smell smell tubes tubes ForFor PAGE SDS PAGE SDS resuspendresuspend samplessamples inin a a minimalminimal volumevolume ofof samplesample buffer buffer TCA DOC AcetoneTCA DOC Acetone UsefulUseful methodmethod toto concentrateconcentrate proteinsproteins andand removeremove acetoneacetone andand TCATCA solublesoluble interferencesinterferences 1 1 ToTo oneone volumevolume ofof proteinprotein solutionsolution addadd 2 2 NaNa deoxycholatedeoxycholate DOC DOC toto 0 02 0 02 finalfinal for for 100100 l l sample sample addadd 1 1 l l 2 2 DOC DOC 2 2 MixMix andand keepkeep atat roomroom temperaturetemperature forfor atat leastleast 1515 min min 3 3 100 100 trichloroacetictrichloroacetic acidacid TCA TCA toto getget 10 10 finalfinal concentrationconcentration preparation preparation ofof 100 100 TCA TCA 454ml454ml H2O kgH2O kg TCA TCA MaintainMaintain inin darkdark bottlebottle atat 4oC Be4oC Be careful careful useuse gloves gloves 4 4 MixMix andand keepkeep atat roomroom temperaturetemperature forfor atat leastleast 1 1 hour hour 5 5 SpinSpin atat 4oC4oC forfor 1010 min min removeremove supernatantsupernatant andand retainretain thethe pellet pellet DryDry tubetube byby inversioninversion onon tissuetissue paper paper 6 6 AddAdd 200200 l l ofof iceice coldcold acetoneacetone toto TCATCA pellet pellet 7 7 MixMix andand keepkeep onon iceice forfor atat leastleast 1515 min min 8 8 SpinSpin atat 4oC4oC forfor 1010 minmin inin microcentrifugemicrocentrifuge atat maximummaximum speed speed 9 9 RemoveRemove supernatantsupernatant asas beforebefore 5 5 drydry airair pelletpellet toto eliminateeliminate anyany acetoneacetone residueresidue smell smell tubes tubes ForFor PAGE SDS PAGE SDS resuspendresuspend samplessamples inin a a minimalminimal volumevolume ofof samplesample buffer buffer 10 10 The The presencepresence ofof somesome TCATCA cancan givegive a a yellowyellow colourcolour asas a a consequenceconsequence ofof thethe acidificationacidification ofof thethe samplesample bufferbuffer titratetitrate withwith 1N1N NaOHNaOH oror 1M1M TrisHClTrisHCl pH8 5pH8 5 toto obtainobtain thethe normalnormal blueblue samplesample bufferbuffer colour colour AcidifiedAcidified Acetone MethanolAcetone Methanol UsefulUseful methodmethod toto removeremove acetoneacetone andand methanolmethanol solublesoluble interferencesinterferences likelike SDSSDS beforebefore IEFIEF 1 1 PreparePrepare acidifiedacidified acetone acetone 120ml120ml acetoneacetone 10 l10 l HClHCl 1mM 1mM finalfinal concentration concentration 2 2 PreparePrepare precipitationprecipitation reagent reagent MixMix equalequal volumesvolumes ofof acidifiedacidified acetoneacetone andand methanolmethanol andand keepkeep atat 20oC 20oC 3 3 ToTo oneone volumevolume ofof proteinprotein solutionsolution addadd 4 4 volumesvolumes ofof coldcold precipitationprecipitation reagent reagent MixMix andand keepkeep ONON atat 20oC 20oC 4 4 SpinSpin 15min15min 4oC4oC inin microfugemicrofuge atat maximummaximum speedspeed 15000g 15000g CarefullyCarefully dischargedischarge supernatantsupernatant andand retainretain thethe pellet pellet drydry tubetube byby inversioninversion onon tissuetissue paperpaper pellet pellet maymay bebe difficultdifficult toto see see 5 5 DryDry samplessamples underunder vaccumvaccum speed vac speed vac oror drydry airair toto eliminateeliminate anyany acetoneacetone oror methanolmethanol residueresidue smell smell tubes tubes TCA EthanolTCA Ethanol PrecipitationPrecipitation UsefulUseful methodmethod toto concentrateconcentrate proteinsproteins andand removalremoval ofof GuanidineGuanidine HydrochlorideHydrochloride beforebefore PAGE SDSPAGE SDS 1 1 DiluteDilute 10 25 l10 25 l samplessamples toto 100 l100 l withwith H2OH2O AddAdd 100 l100 l ofof 20 20 trichloroacetictrichloroacetic acidacid TCA TCA andand mixmix preparation preparation ofof 100 100 TCA TCA 454ml454ml H2O kgH2O kg TCA TCA MaintainMaintain inin darkdark bottlebottle atat 4oC Be4oC Be careful careful useuse gloves gloves 2 2 LeaveLeave inin iceice forfor 20min 20min SpinSpin atat 4oC4oC forfor 1515 minmin inin microcentrifugemicrocentrifuge atat maximummaximum speed speed 3 3 CarefullyCarefully dischargedischarge supernatantsupernatant andand retainretain thethe pellet pellet drydry tubetube byby inversioninversion onon tissuetissue pellet pellet maymay bebe difficultdifficult toto see see 4 4 WashWash pelletpellet withwith 100 l100 l ice coldice cold ethanol ethanol drydry andand resuspendresuspend inin samplesample buffer buffer 5 5 InIn casecase therethere areare tracestraces ofof GuHClGuHCl present present samplessamples shouldshould bebe loadedloaded immediatelyimmediately afterafter boilingboiling forfor 7 7 minmin atat 95 C95 C 6 6 The The presencepresence ofof somesome TCATCA cancan givegive a a yellowyellow colourcolour asas a a consequenceconsequence ofof thethe acidificationacidification ofof thethe samplesample bufferbuffer titratetitrate withwith 1N1N NaOHNaOH oror 1M1M TrisHClTrisHCl pH8 5pH8 5 toto obtainobtain thethe normalnormal blueblue samplesample bufferbuffer colour colour PAGEPAGE prepTMprepTM ProteinProtein Clean upClean up andand EnrichmentEnrichment KitKit PIERCEPIERCE TheThe PAGEPAGE prep prep KitKit enablesenables removalremoval ofof manymany chemicalschemicals thatthat interfereinterfere withwith SDS PAGESDS PAGE analysis analysis guanidine guanidine ammoniumammonium sulfate sulfate otherother commoncommon salts salts acidsacids andand bases bases detergents detergents dyes dyes DNA DNA RNA RNA andand lipids lipids PIERCE PIERCE 26800 26800 PAGEPAGE prepTMprepTM ProteinProtein Clean upClean up andand EnrichmentEnrichment KitKit pdf pdf ChloroformChloroform MethanolMethanol PrecipitationPrecipitation UsefulUseful methodmethod forfor RemovalRemoval ofof saltsalt andand detergentsdetergents 1 1 ToTo samplesample ofof startingstarting volumevolume 100100 ulul 2 2 AddAdd 400400 ulul methanolmethanol 3 3 VortexVortex wellwell 4 4 AddAdd 100100 ulul chloroformchloroform 5 5 VortexVortex 6 6 AddAdd 300300 ulul H2OH2O 7 7 VortexVortex 8 8 SpinSpin 1 1 minuteminute 14 000014 0000 g g 9 9 RemoveRemove toptop aqueousaqueous layerlayer protein protein isis betweenbetween layers layers 10 10 AddAdd 400400 ulul methanolmethanol 11 11 VortexVortex 12 12 SpinSpin 2 2 minutesminutes 14 00014 000 g g 13 13 RemoveRemove asas muchmuch MeOHMeOH asas possiblepossible withoutwithout disturbingdisturbing pelletpellet 14 14 Speed VacSpeed Vac toto drynessdryness 15 15 BringBring upup inin 2X2X samplesample bufferbuffer forfor PAGEPAGE Reference Reference Wessel Wessel D D andand Flugge Flugge U U I I Anal Anal Biochem Biochem 1984 1984 138 138 141 143141 143 哈哈 我做过这个论文哈 1 配胶缓冲液系统对电泳的影响 在 SDS PAGE 不连续电泳中 制胶缓冲液使用的是 Tris HCL 缓冲系 统 浓缩胶是 pH6 7 分离胶 pH8 9 而电泳缓冲液使用的 Tris 甘氨酸缓冲 系统 在浓缩胶中 其 pH 环境呈弱酸性 因此甘氨酸解离很少 其在电场的 作用下 泳动效率低 而 CL 离子却很高 两者之间形成导电性较低的区带 蛋白分子就介于二者之间泳动 由于导电性与电场强度成反比 这一区带便形 成了较高的电压剃度 压着蛋白质分子聚集到一起 浓缩为一狭窄的区带 当 样品进入分离胶后 由于胶中 pH 的增加 呈碱性 甘氨酸大量解离 泳动速 率增加 直接紧随氯离子之后 同时由于分离胶孔径的缩小 在电场的作用下 蛋白分子根据其固有的带电性和分子大小进行分离 所以 pH 对整个反应体系的影响是至关重要的 实验中在排除其他因素 之后仍不能很好解决问题的情况 应首要考虑该因素 2 样品如何处理 根据样品分离目的不同 主要有三种处理方法 还原 SDS 处理 非还原 SDS 处理 带有烷基化作用的还原 SDS 处理 1 还原 SDS 处理 在上样 buffer 中加入 SDS 和 DTT 或 Beta 巯基乙 醇 后 蛋白质构象被解离 电荷被中和 形成 SDS 与蛋白相结合的分子 在 电泳中 只根据分子量来分离 一般电泳均按这种方式处理 样品稀释适当浓 度 加入上样 Buffer 离心 沸水煮 5min 再离心加样 2 带有烷基化作用的还原 SDS 处理 碘乙酸胺的烷基化作用可以很好 的并经久牢固的保护 SH 基团 得到较窄的谱带 另碘乙酸胺可捕集过量的 DTT 而防止银染时的纹理现象 100ul 样品缓冲液中 10ul 20 的碘乙酸胺 并在室温保温 30min 3 非还原 SDS 处理 生理体液 血清 尿素等样品 一般只用 1 SDS 沸水中煮 3min 未加还原剂 因而蛋白折叠未被破坏 不可作为测定 分子量来使用 3 SDS PAGE 电泳凝胶中各主要成分的作用 聚丙烯酰胺的作用 丙烯酰胺与为蛋白质电泳提供载体 其凝固的好坏 直接关系到电泳成功与否 与促凝剂及环境密切相关 制胶缓冲液 浓缩胶选择 pH6 7 分离胶选择 pH8 9 选择 tris HCL 系统 TEMED 与 AP AP 提供自由基 TEMED 是催化剂 催化