ApplicationNotes | Westernblot(seeapplicationnotesbelow).SuggestedblockingbufferisTBS-Tweenwith2%BSA.SuggesteddilutionbufferisTBS-Tweenwith0.05%sodiumazide.Preferredgelpercentageis5%(seeapplicationnotes).
Immunohistochemistryonfrozenandparaffinembeddedtissuesections.Suggestedfixationforfrozentissuesectionsisacetonefixfor6minutesatroomtemperature.Forformalinfixedparaffinembeddedtissuesections:microwavein0.01Mcitratebuffer(pH6.0)for8-10minutes(notethatallmicrowavesdifferandadjustmentsmayneedtobemade)followwithenzymedigestion(0.01%pronasefor10mintues).Suggestedblockingagentisfetalbovineserum.
Immunocytochemistry
Immunoprecipitation.Suggestedextractionbufferis20mMTris-HCl,pH7.4,150mMNaCl,1%TritonX-100,0.1%SDS,0.5%deoxycholicacid-NaCland0.5mMPMSF.Finalreactionvolumeis1mLandsuggestedcaptureagentisagaroseconjugatedanti-mouseIgG.
Optimalworkingdilutionsmustbedeterminedbytheenduser.
APPLICATIONNOTESFORMAB3568
WESTERNBLOT
Toachievegoodresolutionofmyosinheavychainisoformswithdistinctmolecularweight(200-2004kDa),thefollowingprocedureshouldbefollowed:1).Pyrophoshateextractionbufferforsamplepreparation(seebelow);runSDS-PAGEin5%gel.Important:forbetterresolutionoftheMHCbands,useelectrophoreticbufferwithpH8.2(i.e.0.1lessthanstandard),andprepareresolvinggel(5%)withpH9.0(not8.8asusual).Alsohelpthoroughdegasingoftheresolvinggelmixture(H2O,acrylamide,EDTA,pH9.0,before(!)addingSDS,TEMEDandAPS).RunSDS-PAGElongerthanafterthedyefrontrunsoff(use200kDaMWMarkersandletit"s200kDabandrunatleasttothemiddleof8X8gel(usingabigsizegel(notthemini-gel!)willenhancethequalityofMHCbandresolution).
Pyrophosphateextractionbuffer:(40mMNa4P2O7x10H2O,1mMMgCl2,1mMEGTA(addKOHtodissolveEGTA),PMSF,pH9.5).Toextractacto-myosinfromtissues/cells,shakemincedtissueorcellsincoldextractionbuffer1hronicebath(0oC),centrifuge@10,000gfor10minat+2-8oC,takesupernatantandmixit1:1withstandardLaemmlisamplebuffer,boil,runSDS-PAGEin5%gel(seeabove). |