IsolationofProteinfromTissue

1. Placethetissuesonlabeledaluminumfoilandimmediatelyplaceindryice.Itisimperativethatthetissuesstaycoldsothatproteasedonothavetimetoactontheprotein.
2. PlacethetissuesinaroundbottomtubeandaddBrijbufferwithproteaseinhibitorsadded.Addabout1mlBrij/tissuesthatequalsabout100µlinvolume.Brijwithinhibitorsonicebeforeuse.Brij150(Lysisbuffer)

   Tris	1M	1ml
   EDTA	0.5M	0.4ml
   NaCl	5M	3ml
   Brij96	10%	8.75ml
   NP40	10%	1.25ml

   QSto100mlwithH₂O.Proteaseinhibitors(addtotheamountthatyouwillneed).Keepthesolutiononiceafteradditionofinhibitors.

   Leupeptin	1:1000
   Aprotinin	1:1000
   AEBSF	1:1000

   (For10mluse10µlofeachinhibitor.)
3. Usetheblendertodispersetissuesintothebuffer.useforabout5secondsandthenplacethesamplesoniceagaintokeepitfromgettingwarm.WashtheblenderbetweensampleswithPBS.
4. Whensamplesareinsolution(orascloseasyoucangetthem),transferto1.5mlEppendorftubesandcentrifugeinthecoldroomfor10minutesatfullspeed.Removethesupernatantwhichcontainstheproteinandplaceinaneweppendorfonice.
5. DoaproteinassayusingELISAreader.
6. Storeremainderofsampleat-20°Cinanon-frost-freefreezer.Freezingandthawingofproteinsamplesdegradesthem.Labelsampleswithdateorexperimentnumberandproteinconcentration,ifknown.
7. Thawsamplesonice.Remove50µlofprotein,inaneweppendorftube.Freezeremainderofsamples.Bringsamplesupto25µlwithBrijbufferandequalamountof2Xreducingbuffer.AlsomakeatubeofMarkersbyusing10µlofrainbowmarker,15µloflysisbuffer,and25µlofreducingbuffer.Boilsamplesfor5minutesusingatubeholderwhichkeepsthelidsfrompoppingoff.Centrifugebrieflytocollectsampleatbottomoftube.Itisnownotcrucialtokeepthesamplescold.Theyarestableafteradditionofreducingbuffer.Iusuallystorethemoniceanywayuntilreadytoloadonthegel,though.

PreparationofProteinLysatesfromLymphoblastsorFibroblasts

Collectcells(lymphoblastorfibroblast)fromtissuecultureflaskandwash3timeswith1Xphosphatebuffersaline(PBS),pH7.4.Iadd100µlBrijbuffer(forcellscollectedfrom75mmflask).Sonicatethecellpelletsfor45secondsandkeepthepelletonicefor2minutes.Repeatsonicationtwomoretimes.keepthepelletimmediateaftersonicationonice.hereonwardspelletshouldbeonice,otherwiseproteinwillbedegraded,andquicklyso.Afterthreesonications,spinthelysateat14000RPMfor10minutesinthecoldroomusingamicrocentrifuge.Nowtheproteinlysateisreadytoship.Ifyouareshippingoverseaspleaseuseenoughdryiceinyourshippingbox.

Ifyouwanttorunawestern,assaytheproteinusingelisamethodoranyothermethod.Forlongerstorage,pleasestoreproteinlysatesin-80°C.Foranimmediatewestern,takeequalvolumesofproteinlysatesand2Xreducingbuffer(loADIngdye),boilthelysateswithreducingsolutionfor5minutesandthencoolitdown.Spinagaintheproteinlysatefor5minutesat14000RPMinthecoldroomandthenloadonagel.Alwaysuserainbowmarkeroranyotherproteinmarkertoestimatetheproteinsizes.

Proteinlysatebuffer(Brijbuffer):

Makeupto100mlwithddH₂O.

Proteaseinhibitors:

For100mlBrijbufferadd100µlofatleasttwoproteaseinhibitors.

Reducingsolution:

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