沉淀蛋白質(zhì)地常用方法

實(shí)用標(biāo)準(zhǔn)文案沉蛋質(zhì)常方（TCA乙、酮淀白作驟2010-08-1815:19TCA-DOCForprecipitationveryproteinconcentration1)Tooneproteinsolution,add1/100vol.2%DOC(Nadeoxycholate,detergent).2)Vortexandletsit30min4oC.3)Add1/10ofTrichloroacetic(TCA)vortexletON4oC(preparationof100%TCA:454mlH2O/kgMaintaindarkat4oC.Becareful,usegloves!!!).4)Spin15min4oCinmicrofugemaximum(15000g).Carefullydischargesupernatantandthedrytubeinversiontissuepapermaybedifficult[OPTION:Washpellettwicerepellet5minfullbetweenwashes].5)Drysamplesunder(speedordryair.ForSDS,resuspendinaminimalofsamplebuffer.(Thepresenceofsomeayellowcolourasaconsequencetheacidificationofthebuffertitratewith1NNaOHorTrisHClpH8.5toobtainthebluebufferNormalTCAToeliminatesolubleinterferencesandproteinconcentration1)ToasampleproteinsolutionaddTrichloroaceticacid(TCA)100%toget13%finalconcentration.and–20oCandthen15min4oC;longerat4oCthe–steplowerconcentration.Suggestion:leaveONiftheproteinconcentrationisverylow.(preparationof100%454mlTCA.Maintainindarkat4oC.Beusegloves!!!).2)Spin15min4oCinmicrofugemaximum(15000g).Carefullydischargesupernatantandthedrytubeinversiontissue(pelletmaybedifficulttosee).3)SDS,resuspendsamplesinaminimalofbuffer.(TheofsomeTCAcangiveyellowasaconsequenceoftheacidificationthesamplebuffer;titratewith1NorTrisHClpH8.5obtainnormalsamplecolour.)AcetonePrecipitationToeliminateacetonesolubleinterferencesandproteinconcentration精彩文檔

實(shí)用標(biāo)準(zhǔn)文案1)Addvolumeproteinsolutionto4volumescoldacetone.Mixandat20min–20oC.(Suggestion:leaveiftheconcentrationisvery2)Spin15min4oCinmicrofugemaximum(15000g).Carefullydischargesupernatantandthedrytubeinversiontissue(pelletmaybedifficulttosee).3)Drysamplesunder(speed-vac)drytoeliminateacetone(smellForSDS,resuspendinminimalofsamplebuffer.EthanolPrecipitationUsefultoconcentrateproteinsandremovalGuanidineHydrochlorideSDS1)Addtovolumeproteinsolution9volumescold100%.Mixandkeepleast10min.at–20oC.(Suggestion:leaveON).2)Spin15min4oCinmicrocentrifuge(15000g).Carefullydischargesupernatantandthedrytubeinversiontissue(pelletmaybedifficulttosee).3)pellet90%ethanolat–20oC).andrepelletsamplesatfull4)Drysamplesvaccum(speedvac)ordryairtoeliminateanyethanolresiduetubes).SDS,resuspendsamplesaminimalvolumesampleTCA-DOC/AcetoneUsefulmethodtoconcentrateproteinsandacetoneandTCAinterferences1.volumeofproteinadd2%Nadeoxycholate(DOC)0.02%final100μlsample,1μlDOC).2.Mixandkeepattemperatureatleastmin.3.100%trichloroaceticacid(TCA)toget10%finalconcentration(preparationof100%454mlTCA.Maintainindarkat4oC.Beusegloves!!!).4.Mixandkeepattemperatureatleast1hour.5.Spinatfor10min,removesupernatantandretainpellet.Drytubeinversiontissue6.Add200μloficecoldtoTCApellet.7.Mixandkeeponforleastmin.8.Spinat4oCforminmicrocentrifugeatmaximum9.Removesupernatantbeforedryairpellettoeliminateacetone(smellForSDS,resuspendinminimalofsamplebuffer.精彩文檔

實(shí)用標(biāo)準(zhǔn)文案10.presencesomecanayellowcolourasconsequenceoftheacidificationofthesample;titratewithNaOH1MTrisHCltoobtainthenormalbluesamplebuffercolour.)AcidifiedAcetone/MethanolUsefulmethodtoremoveacetonemethanolinterferencesSDSIEF1)Prepareacidifiedacetone:acetone10μlHCl(1mMfinalconcentration).2)Prepareprecipitationreagent:Mixequalvolumesacidifiedandmethanolandat3)Tooneofproteinsolutionadd4volumesprecipitationreagent.andONat-20oC.4)Spin15min4oCinmicrofugemaximum(15000g).Carefullydischargesupernatantandthedrytubeinversiontissue(pelletmaybedifficulttosee).5)Drysamplesunder(speed-vac)drytoeliminateacetonemethanol(smellTCA-EthanolPrecipitationUsefultoconcentrateproteinsandremovalGuanidineHydrochlorideSDS1)Dilute10-25lsamples100lwithH2OAdd100μltrichloroaceticacid(TCA)andmix(preparationTCA:H2O/kgMaintaindarkat4oC.Becareful,usegloves!!!).2)Leaveice20min.at4oCfor15mininmicrocentrifugemaximum3)Carefullydischargesupernatantandretainthedrybyinversionontissuemaybedifficultsee).4)Washpellet100μlice-coldethanol,dryresuspendinbuffer.5)IncasetherearetracesofGuHClpresent,samplesbeimmediatelyboiling7minat6)(ThepresenceofsomeTCAcangiveyellowasaconsequenceoftheacidificationofthesample;titratewithNaOH1MTrisHCltoobtainthenormalbluesamplebuffercolour.)PAGEProteinandEnrichmentKit-PIERCEThePAGEprep?enablesremovalmanychemicalsthatinterferewith精彩文檔

實(shí)用標(biāo)準(zhǔn)文案SDSanalysis:guanidine,ammoniumsulfate,commonacidsandbases,detergents,dyes,DNA,RNA,andPIERCE:-prepTMClean-upEnrichment(pdf)ChloroformPrecipitationUsefulforRemovalofsaltdetergents1)Tosampleofstartingvolume1002)Add400ulmethanol3)Vortexwell4)Add100ulchloroform5)Vortex6)Add300ulH2O7)Vortex8)Spin1minute14,00009)Removetoplayerisbetweenlayers)10)Add400ulmethanol11)12)2minutes@14,000g13)asmuchMeOHaspossiblewithoutdisturbing14)Speed-Vactodryness15)up2XsamplebufferPAGEReference:Wessel,D.andFlugge,U.I.Anal.138,141-143哈哈,我做過這個(gè)論文哈1.配膠緩沖液系統(tǒng)對(duì)電泳的影響？在SDSPAGE連續(xù)電泳中，制膠緩沖液使用的是Tris－HCL沖系統(tǒng)，濃縮膠是，分離膠pH8.9；而電泳緩沖液使用Tris甘氨酸緩沖系統(tǒng)。在濃縮膠中，其環(huán)境呈弱酸性，因此甘氨酸解離很少，其在電場(chǎng)的作用下，泳動(dòng)效率低；而離子卻很高，兩者之間形成導(dǎo)電性較低的區(qū)帶，蛋白分子就介于二者之間泳動(dòng)由于導(dǎo)電性與電場(chǎng)強(qiáng)度成反比這一區(qū)帶便形成了較高的電壓剃度壓著蛋白質(zhì)分子聚集到一起濃縮為一狹窄的區(qū)帶當(dāng)樣品進(jìn)入分離膠后，由于膠中pH的增加，呈堿性，甘氨酸大量解離，泳動(dòng)速率增加，直接緊隨氯離子之后同時(shí)由于分離膠孔徑的縮小在電場(chǎng)的作用下蛋白分子根據(jù)其固有的帶電性和分子大小進(jìn)行分離。所以，pH對(duì)整個(gè)反應(yīng)體系的影響是至關(guān)重要的，實(shí)驗(yàn)中在排除其他因素之后仍不能很好解決問題的情況，應(yīng)首要考慮該因素。2.樣品如何處理？根據(jù)樣品分離目的不同，主要有三種處理方法：還原理、非還原SDS理、帶有烷基化作用的還原SDS理。精彩文檔

實(shí)用標(biāo)準(zhǔn)文案1還原SDS理：在上樣buffer加入SDSDTT或Beta巰基乙醇）后，蛋白質(zhì)構(gòu)象被解離，電荷被中和，形成蛋白相結(jié)合的分子，在電泳中只根據(jù)分子量來分離般電泳均按這種方式處理樣品稀釋適當(dāng)濃度，加入上樣Buffer，離心，沸水煮5min再離心加樣。2帶有烷基化作用的還原SDS理：碘乙酸胺的烷基化作用可以很好的并經(jīng)久牢固的保護(hù)SH基團(tuán)到較窄的譜帶碘乙酸胺捕集過量的，而防止銀染時(shí)的紋理現(xiàn)象。100ul樣品沖液中10ul20％碘乙酸胺，并在室溫保溫30min3非還原SDS處理：生理體液、血清、尿素等樣品，一般只用SDS沸水中煮3min，未加還原劑，因而蛋白折疊未被破壞，不可作為測(cè)定分子量來使用。3.SDS電泳凝膠中各主要成分的作用？聚丙烯酰胺的作用丙烯酰胺與為蛋白質(zhì)電泳提供載體其凝固的好壞直接關(guān)系到電泳成功與否，與促凝劑及環(huán)境密切相關(guān)；制膠緩沖液：濃縮膠選擇pH6.7，分離膠選擇pH8.9，選擇tris－HCL統(tǒng)，與AP：AP提供自由基，TEMED是催化劑，催化自由基引起的聚合反應(yīng)進(jìn)行；十二烷基硫酸鈉SDS）：陽(yáng)離子去污劑，作用有四：去蛋白質(zhì)電荷、解離蛋白質(zhì)之間的氫鍵、取消蛋白分子內(nèi)的疏水作用、去多肽折疊。4.提高SDS泳分辨率的途徑？聚丙烯酰胺的充分聚合可提高凝膠的分辨率建議做法待凝膠在室溫凝固后可在室溫下放置一段時(shí)間使用忌即配即用或冰箱放置前者易導(dǎo)致凝固不充分，后者可導(dǎo)SDS晶。一般凝膠可在室溫下保4，SDS水解聚丙烯酰胺。一般常用的有氨基黑考馬斯亮藍(lán)銀染色三種染料不同染料又各自不同的染色方法，具體可參照郭堯君編著的《蛋白質(zhì)電泳技術(shù)手冊(cè)》P82-103。5.“微笑”（兩邊翹起中間凹下）形帶原因？主要是由于凝膠的中間部分凝固不均勻所致，多出現(xiàn)于較厚的凝膠中。處理辦法：待其充分凝固再作后續(xù)實(shí)驗(yàn)。6.“眉（兩邊向下中間鼓起）形帶原因？主要出現(xiàn)在蛋白質(zhì)垂直電泳槽中般是兩板之間的底部間隙氣泡未排除干凈。處理辦法：可在兩板間加入適量緩沖液，以排除氣泡。7.為什么帶出現(xiàn)拖尾現(xiàn)象？主要是樣品融解效果不佳或分離膠濃度過大引起的。處理辦法：加樣前離心；選擇適當(dāng)?shù)臉悠肪彌_液，加適量樣品促溶劑；電泳緩沖液時(shí)間過長(zhǎng)，重新配制；降低凝膠濃度。8.為什么帶出現(xiàn)紋理現(xiàn)象？主要是樣品不溶性顆粒引起的。處理辦法：加樣前離心；加適量樣品促溶劑。9.什么是“鬼帶，如何處理？鬼帶”就是在跑大子構(gòu)象復(fù)雜的蛋白質(zhì)分子時(shí)，常會(huì)出現(xiàn)在泳道頂端（有時(shí)在濃縮膠中的一些大分子未知條帶或加樣孔底部有沉淀主要由于還