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Howlongshouldcellsbelabeled?Theideallengthoftimetolabelcellsdependsontheproteinofinterestandthelabelthatyouareusing.Ifyouwanttolabelanunstableproteinwith35S-methionine,ashortlabelinginterval--nomorethan2hr--isbest.Ifyouarestudyingastableprotein,alongerlabelmaybepreferable.Theissueisthehalf-lifeoftheproteinofinterestrelativetothehalf-lifeofthebackgroundbands.Thehalf-lifeoftotalcellularproteinis45to50hr. 
Labelingwith32Piisdifferent.Inmostcases,thephosphateinproteinsundergoescontinualturn-over.Therefore,botholdandnewly-synthesizedproteinsbecomelabeledsoonafter32Piisaddedtothecells.Ashortlabelingperiodwith32PiisadvantageousinthatthelabelingofRNAandDNAislessobviousthanincellslabeledover-night.Additionally,forthoseofyoustudyingtyrosineproteinkinases,phosphotyrosinestendtoturn-overfasterthanthebulkofeitherphosphoserineorphosphothreonineandthusarepreferentiallylabeledduringbrieflabeling.If,however,youwanttolookatthesteady-stateabundanceofphosphateinproteins,lipids,orRNA,anover-nightlabelingperiodmaybethebest.Undertheseconditions,youcanbereasonablycertainthatthespecificactivityoftheATPpoolinthecellandofthephosphatesinmacromoleculesisbeginningtoapproachthatofthemedium.Additionally,theamountoflabelpresentinproteins,lipidsandRNAshouldnowreflecttheamountofphosphatepresent,ratherthantherateofturn-overofthephosphate.Anissuetoconsiderhoweveristhefactthatradiationdamagecaninducethestabilizationofp53andcausecellcyclearrest.Cellslabeledforaprolongedperiodwith32Pmaythereforenotbegrowingwhenyouharvestthem.
Manyyearsago,MikeWeberandDocEdlinconcludedthatthespecificactivityoftheATPpoolhadcometoequilibriumwiththephosphateinthemediumby6hours.Theremaythereforebenoreasontolabelforlongerwith32Pi.Overnightlabelingisbestdoneinmediumcontainingareducedconcentrationofphosphateormethionine.10%isoftenreasonable,dependingonthecellline.Thisiseasilyaccomplishedbyusingmethionine-freeorphosphate-freemediumand10%undialyzedserum,whichcanbeassumedtocontainthesameconcentrationofmethionineorphosphateasnormalmedium.Forshorttermlabeling--30secondsto5hrs--use(1)mediumcompletelylackingeitherphosphateormethionine,(2)serumwhichhasbeendialyzedagainstsaline,and(3)afairlylowvolumeofmedium;0.75mlfora35mmdish,2to2.5mlfora50mmdish,and2.5to5mlfora100mmdish.Itisagoodideatorinsethecellsyouaregoingtolabelwithlabelingmedium--whichlackslabel--priortoaddingtheactuallabelingmedium.Starvationdoesnthelpmuch.