Phosphoaminoacidanalysis:MarkKamps"smethod 1.Labelyourproteinwith32Pi.ThensubjecttheproteintoSDSpolyacrylamidegelelectrophoresisandtransferyourgel-fractionatedproteintoImmobilon-P. 2.Cutoutbandofinterest,re-wetinmethanol,rinsewellinwaterandplaceinascrew-captubecontaining150µl5.7NHClorenoughtosubmergeyourpieceofmembrane.3.Incubatein110°Covenfor60min.4.Microfugesamplefullspeedforatleast1min.5.TransfersupernatanttonewtubeandlyophilizeonSpeedivac.(Ittakesabout3hr.)6.ResUSPendinH2O,andmicrofuge5min.Spottingmorethan3µlistedious,sodon"tusemuchH20.Ontheotherhand,Iwouldn"tusemuchlessthan6µlsoastohavegoodrecovery. Ingeneral,youwillneed2litersofeachbuffer.
Neithernitrocellulosenornylonwillwork!
7.Spot1µlPAAstandards(about0.3µgeachofPSer,PThr,andPTyr)onacellulosethinlayerchromatographyplateandthenspotyoursample.Weuse0.1mmEMcelluloseplates
HowdoIwettheplate?Weuseablotterthathasfour,2cm,circularholescutoutofit,oneforeachorigin.Wecuttheholeswithasharpcorkborer.Theblottercanbemadefromagoodgradeofblottingpaper,ortwolayersofWhatmann3MMsewntogether.Itshouldbewet,butnotdripping.Forthefirstdimension,youwettheplatewithpH1.9buffer.
8.ElectrophoreseatpH1.9,1.5KV,20mininthefirstdimension.9.Letplateairdrywell.HowdoIwettheplatefortheseconddimension?UsethreerectangularpiecesofWhatmann3MMthathavebeenwetwithpH3.5buffer.Wetthebottomoftheplatebelowthelowertwoorigins.Keepthepaperatleast1cmawayfromwherethePAAsare.Thenwettheareabetweenthe4origins.Finallywetthetopoftheplate.Theblottersshouldbequitedamp,butnotsoakingwet.
10.Rotate90°counterclockwise.ElectrophoreseatpH3.5,1.6KV,13minintheseconddimension.TheaboveelectrophoresistimesareappropriatewhenyouareusingaSalk/TVL/MBVLtypeelectrophoresisrig.AknockoffofthisissoldbyCBSscientific.Ifyouareusinganotherrig,youwillhavetooptimizeyourelectrophoresistimes.
11.Dryplateinoven.12.Sprayplatewithninhydrinsol"nforseveralseconds.IncubateinovenuntilcanseepurplespotsofPAAstandards.15minshouldbeplenty.13.MarktheplatewithradioactiveinksothatyoucanextrapolatewheretheoriginswereandcanalignthefilmunambigouslywiththeplateandthePAAMarkers.14.Exposetheplatetoflashedfilmwithascreena-70°C.Filmismuchpreferabletothephosphorimagerbecauseitistransparentandisexactlythesamesizeastheplateandthisfacilitatesalignmentofthefilmandtheplate.Intherarecasethatyouneedquantification,youcanusethephosphorimager.15.Afterdevelopingthefilm,tracethelocationsofthePAAstandardsandtheradioactiveinkmarksontoaXeroxtranspancyandsavethisinyournotebook.
Thisiswhataperfectplatelookslike.
pH1.9buffer.88%Formicacid 50ml Aceticacid 156ml H2O 1794ml
Don"tusethe98%formicacidanddon"tadjustthepH.
pH3.5buffer
Pyridine 10ml Aceticacid 100ml 100mmolarEDTA 10ml H2O 1880ml
Don"tbothertoadjustthepH.
TherehavebeenoccasionalproblemswithbadlysmearedPAAsduringelectrophoresisatpH3.5.SomethingseemstoleachoutofthewicksandmakethePAAsrelativelyinsoluble.Tonythinksitisaluminum.Bartthinksthatit"scalcium.Inanycase,thisproblemcanbepreventedbyincluding0.5mMEDTAinthe3.5buffer.
Thistwodimensionaltechniquewasoriginallydeveloped,inlargepartbyTonyHunter,foranalysisofcellextractsorproteinselutedfromgroundupgels.LikeBillWelchbeforehim,MarkKampstiredofgrindingupgelpiecesandfoundthatyoucouldhydrolyzeproteinsboundtofilters.
Ifyouwanttorefertotheoriginalreferencesforthistechnique,cite:
Hunter,T.R.,andSefton,B.M.(1980)TransforminggeneproductofRoussarcomavirusphosphorylatestyrosine.ProcNatlAcadSciUSA198077:1311-1315.[Abstractofthisarticle]
Kamps,M.P.,andSefton,B.M.(1989)Acidandbasehydrolysisofphosphoproteinsboundtoimmobilonfacilitatesanalysisofphosphoaminoacidsingel-fractionatedproteins.AnalBiochem176:22-7.[Abstractofthisarticle]
