蛋白印迹分析Word下载.docx
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凝胶和固相载体被夹在用缓冲溶液浸湿的滤纸之间,通电时间为10分钟~30分钟。
2.湿法:
凝胶和固相载体夹心浸放在转移缓冲溶液中,转移时间可从45分钟延长到过夜进行。
由于湿法的使用弹性更大并且没有明显浪费更多的时间和原料,因此我们在这里只描述湿法的基本操作过程。
对于目的蛋白的识别需要采用能够识别一抗的第二抗体。
该抗体往往是购买的成品,已经被结合或标记了特定的试剂,如辣根过氧化物酶。
这种标记是利用辣根过氧化物酶所催化的一个比色反应,该反应的产物有特定的颜色且固定在固相载体上,容易鉴别。
因此可通过对二抗的识别而识别一抗,进而判断出目标蛋白所在的位置。
其他的识别系统包括碱性磷酸酶系统和125I标记系统。
【实验操作】
⒈.蛋白质的分离
根据目的蛋白的性质,利用电泳方法将其进行分离。
为提高电转移的效率,通常采用SDS/PAGE技术。
分离实验结束后,首先将样品墙的上边缘用小刀去除,然后在胶板的右上角切一个小口以便定位,小心放入转移缓冲溶液中待用。
⒉.电转移
⑴预备PVDF膜
根据胶的大小剪出一片PVDF膜,膜的大小应略微小于胶的大小。
将膜置于甲醇中浸泡1分钟,再移至转移缓冲溶液中待用。
夹心放置顺序
⑵
制作胶膜夹心
在一浅盘中打开转移盒,将一个预先用转移缓冲溶液浸泡过的海绵垫放在转移盒的黑色筛孔板上,在海绵垫的上方放置经转移缓冲溶液浸湿的3MM纸,小心地将胶板放在3MM纸上,并注意排除气泡。
将PVDF膜放在胶的上方同时注意排除气泡,再在膜的上方放上一张同样用转移缓冲溶液浸湿过的3MM纸并赶出气泡,放置另一张浸泡过的海绵垫,关闭转移盒。
将转移盒按照正确的方向放入转移槽中,转移盒的黑色筛孔板贴近转移槽的黑色端,转移盒的白色筛孔板贴近转移槽的白色端,填满转移缓冲溶液同时防止出现气泡。
⑶
电转移
连接电源,在4°
C条件下维持恒压100v,1小时
⒊.免疫检测
⑴膜染色
断开电源,将转移盒从转移槽中移出,将转移盒的各个部分分开。
用镊子将PVDF膜小心放入一个干净的容器中,用TBS缓冲溶液进行短暂清洗,从膜上剪下一条宽约5mm的膜放入另一个干净的容器中。
将这条膜在染色液中浸泡1分钟,然后在脱色液中脱色30分钟,确定蛋白质已经转移到PVDF膜上。
⑵膜的封锁和清洗
对于没有进行染色的膜,首先倒出TBS缓冲溶液,加入3%封闭缓冲溶液,轻轻摇动至少1小时。
倒掉3%封闭缓冲溶液,并用TBS缓冲溶液清洗3次,每次5分钟。
⑶一抗
倒掉TBS缓冲溶液,加入10ml%封闭缓冲溶液及适量的一抗,轻轻摇动1小时以上。
从容器中倒出一抗及封闭缓冲溶液,用TTBS缓冲溶液清洗两次,每次10分钟。
⑷二抗
倒出TTBS缓冲溶液,加入5ml%封闭缓冲溶液及适量的二抗。
轻轻摇动30分钟,倒出二抗及封闭缓冲溶液,用TTBS缓冲溶液清洗两次,每次10分钟。
⑸检测
倒掉TTBS缓冲溶液,并加入显影剂,轻轻摇动PVDF膜,观察显影情况,当能够清晰的看到显色带时,用蒸馏水在30分钟内分三次清洗PVDF膜以终止显色反应的继续进行。
【实验结果】
检查膜上显色结果,蓝紫色带所对应的即是目标蛋白的位置。
【思考题】
⒈蛋白质印迹法的特点是什么?
⒉请说明什么是BSA?
并说明它在本实验中的作用。
⒊请说明二抗在蛋白质印迹法中的生物学功能。
⒋如何保留抗体?
WesternBlotAnalysis
【Purpose】
ComprehendthetheoryofWesternblotting;
understanditsbasicmanipulationandapplication.
【Principle】
WesternblottingisalsocalledImmunoblotting.Itisakindofimmunochemicaltechniqueswhichisusedtodetectaproteinimmobilizedonamatrix.Thetargetproteincanbeinacrudeextractoramorepurifiedpreparationandthemonoclonalorpolyclonalantibodyagainstthisproteinisnecessarytohelpustorecognizetheantigen.
AsintheFigure,solubleantigens(thetargetprotein)maybeseparatedbyelectrophoresisbasedonitsmolecularweight(SDS/PAGE),sizeandcharge(nondenaturatinggelelectrophoresisorisoelectricpoint(isoelectricfocusing).Aftertheseparation,theproteinsaretransferredfromthegeltoaPVDFmembrane.Onceonthemembraneantibodies(firstantibodies)canbeusedtoprobeforthepresenceofparticularproteinbecauseofthespecificallybindingofantigenwithagainstit.Non-specificbindingsitecanbe“blocked”usingothernon-specificproteinsuchasbovineserumalbuminbeforeaddingfirstantibodytoavoidnon-specificbinding.
Proteintransferismostcommonlyaccomplishedbyelectrophoresis,Thisprocedureiscalledelectrophoreticblotting.Thetwocommonelectrophoreticmethodsare:
⒈Semi-dryblotting,inwhichthegelandimmobilizingmatrixaresandwichedbetweenbuffer-wettedfilterpapersthroughwhichacurrentisappliedfor10-30minutes.
⒉Wet(tank)blotting,inwhichthegel-matrixsandwichissubmergedintransferbufferforelectrophoresis,whichmaytakeaslittleas45minutesormaybeallowedtocontinueovernight
Weonlydescribewetblottinghere,sinceitpermitsgreaterflexibilitywithoutbeingsignificantlymoreexpensiveintimeormaterials.
Thedetectionoftargetproteinisusingasecondantibody,whichcanrecognizethefirstantibody.Typically,thesecondantibodyispurchasedalreadyconjugatedtoalabelingagentsuchastheenzymehorseradishperoxidase.Thismarkeristhenvisualizedbyacolorimetricreactioncatalyzedbytheenzymewhichyieldsacoloredproductthatremainsfixedtothemembrane.Thus,itispossibletorecognizefirstantibodythroughrecognizingsecondantibody,andthenidentifythepositionoftargetprotein.Otherdetectionsystemsincludealkalinephosphataseand125Ilabels.
【Materials】
⒈Apparatus:
ApparatusofSDS-PAGE,ElectroblottingApparatus,Powersupply,PVDFmembrane(MilliporeImmobion-P#IPVH00010),Whatman3MMpaper,AdditionalTools:
Forceps,spongepad,scissor,gloves,smallplasticorglasscontainer,Shallowtray.
⒉Reagents:
⑴10xtransferbuffer(1L):
gTrizmabaseM),144gGlycineM),pHshouldbe;
withoutadjustment.
⑵1xtransferbuffer(2L):
400mlMethanol,200ml10xtransferbuffer,1400mlwater.
⑶TBSbuffer:
AddTris(10mM)andNaCl(150mM)to1LdistilledwaterandadjustpHtowithHCl.
⑷TTBSbuffer:
1LTBSbufferaddTween20%).
⑸Firstantibody:
antibodyagainstthetargetprotein.
⑹Secondantibody:
goatanti-rabbit-HRP(horseradishperoxidase).
⑺3%BlockingbufferL):
Add15mgBovineserumalbumininTBSbuffertofinalvolumeL,keepat4°
Ctopreventbacterialcontamination.
⑻%BlockingbufferL):
AddBovineserumalbumininTBSbuffertofinalL,keepat4°
Ctopreventbacterialcontamination.
⑼Developingreagent:
1mlchlonoaphtholsolution(30mg/mlinmethanol),add10mlmethanol,addTBSbufferto50mlandadd30ul30%H2O2.
⑽Stainingbuffer:
Add1gamidoblack18B%),250mlisopropanol(25%)and100mlaceticacid(10%)todistilledwaterwithfinalvolume1L.
⑾Destainingbuffer:
Add350mlisopropanol(35%)and2mlaceticacid(2%)todistilledwaterwithfinalvolume1L.
【Procedure】
⒈.SeparationofProtein
Runanelectrophoreticseparationofknownantigenicproteins.Themethodofseparationdecidedbythecharactersoftargetprotein,butforsufficientlytransferring,themostcommonmethodisSDS-PAGE.
Afterseparation,removeuppersideofsamplewellswitharazorblade.Notchingbottomright-handcornerofgelfororientationandputgelintransferbufferuntilreadytouse.
⒉.Electrotransfer
⑴Preparationofmembrane
CutapieceofPVDFmembrane(MilliporeImmobion-P#IPVH00010)accordingtothesizeofgel.Incubateinmethanolforabout1minonarockeratroomtemp.Removemethanolandequilibratemembranein1xtransferbufferuntilreadytouse.
⑵Arrangegel-membranesandwich
Inashallowtray,openthetransfercassette.Putawell-soakedspongepadontheblackpieceofthetransfercassetteandawetted3MMpaperonthespongepad.Placethegelonthepaperandarrangewellsothatallairbubblesareremoved.LaythePVDFmembraneonthetopofgelandremoveanyairbubbles.Placeawettedsheetof3MMpaperoverthePVDFmembraneandremovethebubble.Coveredwiththesecondwell-soakedpad.Closethesandwichwiththewhitepieceofthecassette.Mountthesandwichinthetransfertank;
puttheblacksidesneartheblacksideofthedevice.Fillthebuffertankwiththetransferbuffer.
⑶Electrotransfer:
Attachtheelectrodes.Setthepowersupplyto100V(constantvoltage)for1hat4°
C.
⒊.Immunodetection
⑴Membranestaining
Disconnecttransferapparatus,removetransfercassette,andpeel3MMpaperfrommembrane.Removethemembranetoasmallcontainer.Add10mlTBSbufferandwashforshorttime.Cutoutonestripewith5mmwidthandputinanothercleancontainer.Stainthisstripeinstainingbufferfor1min.Destainfor30minindestainingbuffertocheckwhetherproteinhasbeentransferredfromgeltomembraneornot.
⑵Membraneblockingandwashing
Forotherpartofmembrane,pouroffTBSbuffer.Add3%blockingbuffer,rockgentlyforatleast1h.Pouroff3%blockingbufferandrinsebrieflywithTBSbufferthreetimes,5minutesforpertime.
⑶Firstantibody
PouroffTBSbuffer.Addfirstantibodyatappropriatedilutionin10ml%blockingbuffer.Rockgentlyforatleast1h;
pourofffirstantibodysolutionfrommembraneandwashtwicefor10minuteswithTTBSbuffer.
⑷Secondantibody
PouroffTTBSbuffer.Addsecondantibodyatappropriatedilutionin5ml%blockingbuffer.Rockgentlyfor30min,pouroffsecondantibodysolutionfrommembraneandwashtwicefor10minuteswithTTBSbuffer.
⑸Detection
PouroffTTBSbufferfrommembraneandadddevelopingreagent,RockPVDFgently,monitoringdevelopment.Whenthebandscanbeseenclearly,stopdevelopmentbywashingmembranewithdistilledwaterfor30minuteswith3changes.
【Result】
Checkthebandsonmembrane,thebandwithblue-purplecolorcorrespondingtothetargetprotein.
【Questions】
⒈Whatisthecharacterofwesternblotting?
⒉WhatistheBSA?
Andexplainitsfunctionduringthisexperiment.
⒊Pleaseelucidatethefunctionofsecondantibodyduringwesternblotting.
⒋Howtoreserveantibody?