Western blot troubleshooting tips
来自 : 蚂蚁淘
TroubleshootingTipsforWesternImmunoblotting
ThefollowingtipscanbeusedtoovercomethemostcommonproblemsencounteredduringWesternblotting.
SmearedPatternorDistortedBands
- Unevencontactbetweengelandmembrane:cassettesusedshouldallowatightfit,leADIngtoevenpressureovertheentiresurfaceofthegelandmembrane.
- Gelnotequilibratedinbufferpriortotransfer:thegelshouldbesoakedintransferbuffercontainingmethanolfor15to30minutes,intransferbufferwithoutmethanolfor60minutesbeforeassemblingthetransfersandwich.
"BaldSpots"
- Bubblesbetweengelandmembrane:bubblescreatepointsofhighresistancethatleadtolowtransferefficiency,thereforecareshouldbetakentoremovebubblescompletelywhenputtingtogetherthetransfersandwich.
IncompleteTransfer
Oneofseveraltechnicalerrorscanbethesourceofthisproblem:
- Proteinsnotcompletelyelutedoutofgel:thisoftenoccurswithhighmolecularweightproteins,especiallywhenusingatransferbuffercontainingmethanol.Onewaytoovercomethisphenomenonisbyusinganylonmembrane,whichdoesnotrequiremethanolinthetransferbuffer.AddingSDStothetransferbufferaswellasusinghigherfieldstrengthsalsoimprovesproteinelution.
- Proteinshavetransferredthroughmembrane:thismayoccurwhenworkingwithproteinsofverylowmolecularweight.Optimizing/shorteningtransfertimesandusingadoublelayerofmembraneusuallyenablesretentionofsmallproteins.
- Inappropriatetransferbufferused:themoststableandcommonlyusedbuffersareTris-Glycinebased.
- Impuritiesinthetransferbuffer:thiswillleadtoapatternonthemembranethatmirrorstheholesinthetransfercassette.Freshbuffershouldbepreparedpriortoeachtransferprocess.
HighBackground
- Cross-reactivitybetweenblockingagentandprimaryorsecondaryantibody:thiswillresultinanoverallmembranestaining.Usually,theadditionofamilddetergentsuchasTween-20totheincubationandwashingbufferswilleliminatetheproblem.Ifbackgroundstainingpersists,changingtheblockingagentisrecommended.
- Concentrationofantibodytoohighorincubationtimetoolong:thehighertheantibodyconcentrationandthelongertheincubationtime,thegreaterthelikelihoodfornon-specificstaining.Raisingtheincubationtemperature(e.g.to37�C)isrecommendedoverlengtheningtheincubationtime.Also,severalshortwashingstepsarebetterthanonelongone.
- Membranedryduringincubationprocess:careshouldbetakentokeepmembranefromdryingoutduringincubation.
LittleorNoSignal
- Antigenisnotrecognizedbyprimaryantibody:thiscanoccurespeciallywithmonoclonalantibodiesthatwereraisedagainstanativeprotein.Insomecases,anon-reducinggelsystemmayneedtobeused.
- Inhibitionofsecondaryantibodyconjugate:Horseradishperoxidaselabeledantibodiesshouldnotbeusedinconjunctionwithsodiumazideorhemoglobin.Biotinylatedantibodiesshouldnotbeusedwithmilkorcaseinsincebothcontainbiotin.Concanavalinlabeledantibodiesshouldnotbebroughtintocontactwithmilkduetointerferingglycoproteins.Achangeintheblockingagentorincubationsolutionwillsolvethisproblem.
- Detergentistooharsh:SDS,NonidetP-40,andTritonX-100disruptbindingbetweenproteins.Tween-20isthemostcommonlyusedandrecommendeddetergentforwashingandincubationsolutions.
免责声明 本文仅代表作者个人观点，与本网无关。其创作性以及文中陈述文字和内容未经本站证实，对本文以及其中全部或者部分内容、文字的真实性、完整性、及时性本站不做任何保证或承诺，请读者仅作参考，并请自行核实相关内容。
版权声明 未经蚂蚁淘授权不得转载、摘编或利用其他方式使用上述作品。已经经本网授权使用作品的，应该授权范围内使用，并注明“来源：蚂蚁淘”。违反上述声明者，本网将追究其相关法律责任。
