ChIPresultsobtainedwiththemonoclonalantibodydirectedagainstPolII
ChIPassayswereperformedusinghumanHeLacells,themonoclonalantibodyagainstPolII(Cat.#25309)andoptimizedPCRprimerpairsforqPCR.ChIPwasperformedusingshearedchromatinfrom1millioncells.Atitrationconsistingof1,2,5and10µgofantibodyperChIPexperimentwasanalyzed.IgG(2µg/IP)wasusedasanegativeIPcontrol.QuantitativePCRwasperformedwithprimersspecificforthepromoterandthecodingregionoftheconstitutivelyexpressedGAPDHandACTBgenes,usedaspositivecontrols,andforexon2oftheinactivemyoglobin(MB)geneandtheSat2satelliterepeat,usedasnegativecontrols.Figure1showstherecovery,expressedasa%ofinput(therelativeamountofimmunoprecipitatedDNAcomparedtoinputDNAafterqPCRanalysis).

ChIP-seqresultsobtainedwiththemonoclonalantibodydirectedagainstPolII
ChIPwasperformedonshearedchromatinfrom1millionHeLaS3cellsusing1µgoftheantibodyagainstPolII(Cat.#25309)asdescribedabove.TheimmunoprecipitatedDNAwassubsequentlyanalyzedonanIlluminaGenomeAnalyzer.Librarypreparation,clustergenerationandsequencingwereperformedaccordingtothemanufacturer"sinstructions.The36bptagswerealignedtothehumangenomeusingtheELANDalgorithm.Figure2showsthepeakdistributionalongthecompletesequenceanda400kbregionoftheX-chromosome(figure2AandB,respectively),andinatwogenomicregionssurroundingtheGAPDHandACTBpositivecontrolgenes(figure2CandD).

CrossreactivityofthemonoclonalantibodydirectedagainstPolII
TotestthespecificityanELISAwasperformedusingaserialdilutionofthemonoclonalantibodyagainstPolII(Cat.#25309).ThewellswerecoatedwithpeptidescontainingtheunmodifiedC-terminalrepeatsequenceaswellasdifferentphosphorylatedpeptides.Figure3showsthattheantibodyrecognizestheunphosphorylatedPolIIaswellasmostphosphorylatedforms.

WesternblotanalysisusingthemonoclonalantibodydirectedagainstPolII
Nuclearextracts(25µg)fromHeLacellswereanalyzedbyWesternblotusingthemonoclonalantibodyagainstPolII(Cat.#25309)diluted1:1,000inTBS-Tweencontaining5%skimmedmilk.Thepositionoftheproteinofinterestisindicatedontheright;theMarker(inkDa)isshownontheleft.

ImmunofluorescenceusingthemonoclonalantibodydirectedagainstPolII
HeLacellswerestainedwiththeantibodyagainstPolII(Cat.#25309)andwithDAPI.CellswerefixedwithmethanolandblockedwithPBS/TX-100containing5%normalgoatserumand1%BSA.ThecellswereimmunofluorescentlylabelledwiththePolIIantibody(left)diluted1:500inblockingsolutionfollowedbyananti-mouseantibodyconjugatedtoAlexa594.ThemiddlepanelshowsstainingofthenucleiwithDAPI.Amergeofthetwostainingsisshownontheright.

Storeat–80°Cforupto2years.Centrifugeafterfirstthawtomaximizeproductrecovery.Aliquottoavoidrepeatedfreeze/thawcycles.Aliquotsmaybestoredat–20°Cforatleastonemonth.