Isolation of Protein from Tissue
来自 : 蚂蚁淘
IsolationofProteinfromTissue
- Placethetissuesonlabeledaluminumfoilandimmediatelyplaceindryice.Itisimperativethatthetissuesstaycoldsothatproteasedonothavetimetoactontheprotein.
- 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.) - Usetheblendertodispersetissuesintothebuffer.useforabout5secondsandthenplacethesamplesoniceagaintokeepitfromgettingwarm.WashtheblenderbetweensampleswithPBS.
- Whensamplesareinsolution(orascloseasyoucangetthem),transferto1.5mlEppendorftubesandcentrifugeinthecoldroomfor10minutesatfullspeed.Removethesupernatantwhichcontainstheproteinandplaceinaneweppendorfonice.
- DoaproteinassayusingELISAreader.
- Storeremainderofsampleat-20°Cinanon-frost-freefreezer.Freezingandthawingofproteinsamplesdegradesthem.Labelsampleswithdateorexperimentnumberandproteinconcentration,ifknown.
- 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):
| Tris | 1M | 1ml |
| EDTA | 0.5M | 0.4ml |
| NaCl | 5M | 3ml |
| Brij96 | 10% | 8.75ml |
| NP40 | 10% | 1.25ml |
Makeupto100mlwithddH2O.
Proteaseinhibitors:
| Leupeptin | 25µM |
| Aprotinin | 25µM |
| AEBSF | 25µM |
For100mlBrijbufferadd100µlofatleasttwoproteaseinhibitors.
Reducingsolution:
| Bromophenolblue | pinch |
| 0.5MTrispH6.8 | 2.5ml |
| Glycerol | 2.0ml |
| 10%SDS | 2.0ml |
| ddH2O | 2.5ml |
| Beta-mercaptoethanol | 1ml |
| TOTAL | 10ml |
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