Hattoti"sprotocoladaptedtocellculture:HattoriM,TugoresA,VelozL,KarinM,BrennerD(1990)Asimplifiedmethodforthepreparationoftranscrip-tionallyactivelivernuclearextracts.DNACellBiol.Vol.10,P.777–781 I.Homogenizecellsortissue 1.WashcommerciallyavailableHeLacells(30g)ormincedin3mmpiecesfreshliver(15-20g)fromstainingwithhomogenizingbuffer(10ml)andtransferitto100mlbeaker 2.Addtothebeakeranother25mlofthebufferandsplitthecontentinhalf(2x15ml) 3.Homogenizeeachportiontwice,10strokes(“up”and“down”)eachwithateflonpestle.Betweenstrokes,coolhomogenizeronice.Trytomaintaintemperaturecloseto+4C II.Pelletthenuclei 1.Filterhomogenatethroughthefour-layercloth(25-30ml)andmixhomogenatewithcushionbuffer(50mlofcushionbufferper25mlofthehomogenate 2.Distributetoultracentrifugetubescushionbuffer(10mlofbufferperatube).Carefullyloadcushionbuffer-homogenatemixonthetop(totalvolumeisabout38ml). 3EquilibratetubesatthebalanceandcentrifugetheminSW28rotor(24,000rpmor76220g,50min,1C)or60Tirotor(sametimeandtemperatureat32,750rpm/min) III.Lysenuclei 1.Inverttubesandkeepthemonicefor10min 2.ResUSPendpelletsinnuclearlysisbuffer(5ml/pellet).PerformonestrokewithDouncehomogenizer 3Addlysisbufferto40mland2mlof4Mammoniumsulfateandagitatefor30-60minat+4C IV.Reprecipitatenuclearproteins 1.Centrifugelysatesin55.2Tirotor(35,000rpm,60min,+1C)or60Tirotor(37,700rpm,60min,+1C).Savesuper. 2.Addammoniumsulfate(330mg/mlofsolidsaltor65mlof4Msolution)andkeepsample(s)onicefor15min 3Centrifugesample(s)in55.2Tirotoror60Tirotorfor20minatthesamespeedandtemperature.SaveTHEPELLET(S). V.Dialyzenuclearextractandmeasureprotein 1.Resuspendeachpelletin500mlofdialysisbufferanddializesample(s)againstdialysisbuffer(1:1,000for4h) 2Clarifynuclearextractbycentrifugation(+4C,10min)inmicrofuge. 3.Measureproteinandaliquotsupernatant(100-500mg/tube)andstoreitat-70CLargescalenuclearextractpreparation
