Largescalenuclearextractpreparation

Hattoti"sprotocoladaptedtocellculture:HattoriM,TugoresA,VelozL,KarinM,BrennerD(1990)Asimplifiedmethodforthepreparationoftranscrip-tionallyactivelivernuclearextracts.DNACellBiol.Vol.10,P.777–781

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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-70C

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Largescalenuclearextractpreparation

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