ChIPresultsobtainedwiththemonoclonalantibodyagainstTBP
ChIPassayswereperformedusingU2OScells,theantibodydirectedagainstTBP(Cat.#25319)andoptimizedprimersetsforqPCR.Shearedchromatinfrom1x106cellsand4µgofantibodywereusedperChIPexperiment.QPCRwasperformedwithprimersforthepromoterofthec-fosandGAPDHgenes,aregion0.5and1kbupstreamoftheGAPDHpromoter,respectively,andforexon2ofthemyoglobingeneasanegativecontrol.Figure1showstherecovery(therelativeamountofimmunoprecipitatedDNAcomparedtoinputDNA)andtheoccupancy(ratio+/-controltarget).TheseresultsdemonstratetheoccupancyofbothpromotersbyTBP.
ChIP-seqresultsobtainedwiththemonoclonalantibodydirectedagainstTBP
ChIPwasperformedwith5µgoftheantibodyagainstTBP(Cat.#25319)onshearedchromatinfrom1millionHeLaS3cells.TheimmunoprecipitatedDNAwasanalyzedbyQPCRwithoptimizedPCRprimerpairsforthepromotersoftheactiveGAPDHandc-fosgenes,usedaspositivecontroltargets,andforaregion1kbupstreamoftheGAPDHpromoterandthecodingregionoftheinactiveMBgene,usedasnegativecontroltargets(figure2A).TheimmunoprecipitatedDNAwassubsequentlyanalyzedwithanIlluminaGenomeAnalyzer.Librarypreparation,clustergenerationandsequencingwereperformedaccordingtothemanufacturer"sinstructions.The36bptagswerealignedtothehumangenomeusingtheELANDalgorithm.Figure2showsthepeakdistributionin50kbregionssurroundingtheGAPDH,c-fos,ACTBandMCL1genes(figure2B,C,DandE,respectively).TheseresultsclearlyshowalocalisationofTBPatthepromotersofactivelytranscribedgenes.
Storeat–80°Cforupto2years.Centrifugeafterfirstthawtomaximizeproductrecovery.Aliquottoavoidrepeatedfreeze/thawcycles.Aliquotsmaybestoredat–20°Cforatleastonemonth.
WB(1:200–1:2000)
