Phosphoaminoacidanalysis:MarkKamps"smethod

1.Labelyourproteinwith32Pi.ThensubjecttheproteintoSDSpolyacrylamidegelelectrophoresisandtransferyourgel-fractionatedproteintoImmobilon-P.

Neithernitrocellulosenornylonwillwork!

• KeepthemembranewetandwrapinSaranwrap.
• AddrADIoactivemarkingsfororientationoffilmlater
• Exposetofilm.

2.Cutoutbandofinterest,re-wetinmethanol,rinsewellinwaterandplaceinascrew-captubecontaining150µl5.7NHClorenoughtosubmergeyourpieceofmembrane.3.Incubatein110°Covenfor60min.4.Microfugesamplefullspeedforatleast1min.5.TransfersupernatanttonewtubeandlyophilizeonSpeedivac.(Ittakesabout3hr.)6.ResUSPendinH2O,andmicrofuge5min.Spottingmorethan3µlistedious,sodon"tusemuchH20.Ontheotherhand,Iwouldn"tusemuchlessthan6µlsoastohavegoodrecovery.7.Spot1µlPAAstandards(about0.3µgeachofPSer,PThr,andPTyr)onacellulosethinlayerchromatographyplateandthenspotyoursample.Weuse0.1mmEMcelluloseplates

• Youcanspotthewholesampleifyouareskillfulandhavenochoicebecauseyoudon"thavemanycounts.Spot0.25-0.30µlatatimeanddrywithhouseair,blownthroughapluggedpipet,betweenapplications.

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HowdoIwettheplate?Weuseablotterthathasfour,2cm,circularholescutoutofit,oneforeachorigin.Wecuttheholeswithasharpcorkborer.Theblottercanbemadefromagoodgradeofblottingpaper,ortwolayersofWhatmann3MMsewntogether.Itshouldbewet,butnotdripping.Forthefirstdimension,youwettheplatewithpH1.9buffer.

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8.ElectrophoreseatpH1.9,1.5KV,20mininthefirstdimension.9.Letplateairdrywell.

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HowdoIwettheplatefortheseconddimension?UsethreerectangularpiecesofWhatmann3MMthathavebeenwetwithpH3.5buffer.Wetthebottomoftheplatebelowthelowertwoorigins.Keepthepaperatleast1cmawayfromwherethePAAsare.Thenwettheareabetweenthe4origins.Finallywetthetopoftheplate.Theblottersshouldbequitedamp,butnotsoakingwet.

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10.Rotate90°counterclockwise.ElectrophoreseatpH3.5,1.6KV,13minintheseconddimension.

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TheaboveelectrophoresistimesareappropriatewhenyouareusingaSalk/TVL/MBVLtypeelectrophoresisrig.AknockoffofthisissoldbyCBSscientific.Ifyouareusinganotherrig,youwillhavetooptimizeyourelectrophoresistimes.

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11.Dryplateinoven.12.Sprayplatewithninhydrinsol"nforseveralseconds.IncubateinovenuntilcanseepurplespotsofPAAstandards.15minshouldbeplenty.13.MarktheplatewithradioactiveinksothatyoucanextrapolatewheretheoriginswereandcanalignthefilmunambigouslywiththeplateandthePAAMarkers.14.Exposetheplatetoflashedfilmwithascreena-70°C.Filmismuchpreferabletothephosphorimagerbecauseitistransparentandisexactlythesamesizeastheplateandthisfacilitatesalignmentofthefilmandtheplate.Intherarecasethatyouneedquantification,youcanusethephosphorimager.15.Afterdevelopingthefilm,tracethelocationsofthePAAstandardsandtheradioactiveinkmarksontoaXeroxtranspancyandsavethisinyournotebook.

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Thisiswhataperfectplatelookslike.

Ingeneral,youwillneed2litersofeachbuffer.

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pH1.9buffer.

88%Formicacid	50ml
Aceticacid	156ml
H2O	1794ml

Don"tusethe98%formicacidanddon"tadjustthepH.

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pH3.5buffer

Pyridine	10ml
Aceticacid	100ml
100mmolarEDTA	10ml
H2O	1880ml

Don"tbothertoadjustthepH.

TherehavebeenoccasionalproblemswithbadlysmearedPAAsduringelectrophoresisatpH3.5.SomethingseemstoleachoutofthewicksandmakethePAAsrelativelyinsoluble.Tonythinksitisaluminum.Bartthinksthatit"scalcium.Inanycase,thisproblemcanbepreventedbyincluding0.5mMEDTAinthe3.5buffer.

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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]

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