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ActinCaptureAssay
DavidAmberg
- DialyzepurifiedGSTfusionproteinsandactinintoPBS+1mMMgCl2.
- Mix5ugactininto50ultotalvolumebindingbuffer.
- Mix5ugGST-fusionproteinintototalvolume50ulbindingbuffer.
- Combineactin+GST-fusionproteinandincubatefor1hr.x4¡C.
- Add20ul50%glutathioneagaroseequilibratedintobindingbuffer.Incubate15minx4¡Cwithfrequentmixing.
- Washbeads4timeswithbindingbuffer,spindown4-8000rpmfor30seconds.Onthelastwashtransfertoanewtube,spindownandremoveasmuchliquidaspossIBLe.
- Add50ulreducingsamplebuffer,heat2min.x95¡Candload10ulontoa10%SDS-PAGE.
- Executewesternaccordingtomyprotocol.Forprimaryantibodyuseanti-GSTat1:1000andanti-actinat1:1000.ForsecondaryuseHRPconjugatedIgGat1:10,000.
ActinCaptureAssayReagants
- BindingBuffer:1XPBSpH7.2(LaneRecipe)+10%Glycerol+1mMMgCl2+0.2mMATP+0.1%BSA+0.01%TritonX-100+1mMDTT+1mMEGTA.Variablesincludeloweringsalt(aboverecipeisabout136mMinNaCl).replacingEGTAwith1mMCaCl2,replacingATPwithADP.
- ATPSigma#A5394
- ADPSigma#A6521
- 10XPBS:80gramsNaCl+2gramsKCl+14.4gramsNa2HPO4+2.4gramsKH2PO4.pHupondilutionto1Xshouldbe7.2.
- BSASigma#A7638.
- Glutathione-AgaroseSigma#G-4510.Prepareaccordingtoinstructions.
- Anti-GSTMolecularprobes,Inc.#A5800.RabbitIgG(H+L)fraction.
- PeroxidaseconjugatedProteinACappel#55901.
- GuineaPiganti-actinAnimal#2wholeserum,Botsteinlab,preparedbyJonMulholland.
- SampleBuffer:1ml0.5MTRISpH6.8+4.4mlddH2O+2.0ml10%SDS+2.0mlglycerol+1.0ml1MDTT.
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