【官网】中国细胞系_原代细胞_病毒包装_质粒购买_载体 ...
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RunningProteinGels
10XRunningBuffer(0.25MTris,1.92Mglycine,1%SDS)121gTris577gglycine40gSDSddh20to4L(checkpHat1:10dilution(pH=~8.8)).5XSampleBuffer(0.3125MTrispH6.8,10%SDS,50%glycerol,0.005%bromophenolblue,25%2-mercaptoethanol)1.56ml2MTrispH6.81gSDS5mlglycerol0.2ml0.25%solutionofbromophenolblue2.5ml2-mercaptoethanolddH20to10ml.30:0.8Acrylamide:bis0.5MTrispH6.810%SDS10%APSTEMED3MTrispH8.8
| 5%StackingGel |
| 15cmx17cm | 5cmx9cm | Solution |
| 4.2ml | 2.5ml | Acrylamide:bis |
| 5.8ml | 3.5ml | 0.5MTrispH6.8 |
| 15ml | 8.8ml | ddH20 |
| 120µl | 72µl | 10%SDS |
| 120µl | 72µl | 10%APS |
| 50µl | 30µl | TEMED |
| 10%ResolvingGel |
| 15cmx17cm | 5cmx9cm | Solution |
| 15.9ml | 6.7ml | Acrylamide:bis |
| 12ml | 7.5ml | 3MTrispH8.8 |
| 18.9ml | 5.4ml | ddH20 |
| 480µl | 200µl | 10%SDS |
| 480µl | 200µl | 10%APS |
| 96µl | 40µl | TEMED |
StainingProtocols
ModifiedfromHoeferProteinElectrophoresisApplicationsGuide
StainingSolution(0.025%CoomassieBrilliantblueR250,40%methanol,7%aceticacid)0.5gCoomassieBrilliantblueR800mlmethanolStiruntildissolved.Thenadd:140mlaceticacidddH20to2LStoreatroomtemperatureforupto6months.DestainingSolutionI(40%methanol,7%aceticacid)400mlmethanol70mlaceticacidddH20to1LStoreatroomtemperatureadinfinitum.DestainingSolutionII(7%aceticacid,5%methanol)700mlaceticacid500mlmethanolddH20to10LStoreatroomtemperatureadinfinitum.
- PlacegelinStainingSolution.Shakeslowlyfor1hrtoovernight.
- ReplacetheStainingSolutionwithDestainingSolutionI.Shakeslowlyfor30minutes.
- RemoveDestainingSolutionIandreplacewithDestainingSolutionII.AdditionofKimwipestoonecornerofthestainingtraywillhelpremoveCoomassiebluefromthegelwithoutchangingthedestainingsolution.ReplacetissueswhentheyaresaturatedwithCoomassieblue.
- Tominimizecracking,add1%glyceroltothelastdestainbeforedryingthegel.
- Placegelinstainingsolutioninasmallboxwithalid.(Anemptypipettipboxworkswell.)
- Microwaveonhighfor1min,thenshake10-20min.
- ReplacestainingsolutionwithDestainI;AddsomeKimwipesorafolded-uppapertoweltohelpabsorbthestain.
- Microwaveonhighfor1minandshakeuntilthebandsemergeclearlyfromthebackground.
- PouroutDestainI,replacewithwater.Thegelwillcontinuetodestainalittlebit.
- Drydownthegelwhenyougetaroundtoit.
- Fix:50%methanol,10%aceticacid(100ml).
- Microwaveonhighfor1min,thenshake15min.
- Wash:dH20(100ml).
- Microwaveonhighfor1min,thenshake10min(oruntilthegelisrehydrated).
- Reduce:5µg/mlDTTindH20(100mls)(i.e.32.5µlof0.1MDTTinto100mls).
- Microwaveonhighfor1min,thenshake15min(oruntilthegelhascooled).
- Stain:0.1%AgNO3indH20(100ml)(Dilutea10%AgNO3stock).
- DONOTMICROWAVE.Shake15min.
- Develop:Makefreshdevelopersolutionforeachgel(200ml:40ml15%Na2CO3+160mldH20).
- WashquicklywithdH20--2X.Shakewhilewashing.
- Washquicklywithdeveloper--2X50mls.
- Add100µlof37%formaldehydetotheremaining100mlsofdeveloper.
- Pourdeveloperongelandshakeuntilbandsareseen.
- STOPbyadding5mlsof2.3Mcitricacid(shouldbebubbling).
- ShakeinSTOPfor10min,thenwashoutwithdH20severaltimes(otherwisethebackgroundturnsyellow).
- Soakin5%glycerolfor15minorlongerbeforedryinggel.
Ifpreparingasampleformassspectrometry,youmaywishtousetheprotocolfoundhere.
Westerns
- Optional:soakthegelinsemidrytransferbuffer(48mMTris,39mMglycine,0.037%SDS,20%methanol)10-20’.
- Cutthemembrane(Immobilon-P/PVDF)and6piecesofblottingpaper(e.g.SchleicherandSchuellGB002)tothesamesizeasthegel(~8.5x5.7cm).Wetthemembranefor15secondsin100%methanol,thensoakfor2’inwater,thenequilibratethemembranefor5minutesintransferbuffer.
- Assemblethetransferstack:
- Threepiecesofblotpapersoakedintransferbuffer
- Gel
- Pre-soakedmembrane
- Threepiecesofblotpapersoakedintransferbuffer
Placestackupsidedown(gelsideup)onblotter.Blotat0.8mA/cm2(~38mApergel)for1-2hrs.Placethemembraneonapieceofblotpaperanddryfor2hrs(orlonger)atroomtemporsoakthemembranein100%methanolfor10sec.anddry15min.- Wetmembrane5sec.in100%methanol,thenincubatebrieflyinTBST(25mMTris,140mMNaCl,3mMKCl,0.05%Tween-20,pH8.0).
- StainwithIndiaInk(Pelikan,FountIndiaBlack)0.1%inTBSTwith0.02%azide(saveandstoreat4°C).
- Wash2timeswithTBST.
- Blockfor1hratRTorO/Nat4°Cwith5%nonfatdrymilkinTBSTand0.02%azide(saveandstoreat4°C).
- WashbrieflywithTBST.
- IncubatewithfirstantibodyinTBST,2%milk,0.02%azide(saveandstoreat4°C)for1-2hratRT(orO/Nat4°C).
- QuickwashwithTBST,thenwash3x10’withTBST.
- Incubatewithsecondaryantibody(1:5000dilution)inTBST,2%milk(noazide!)for20-60’.
- QuickwashwithTBST,thenwash3x10’withTBST.
- PrepareECLreagents:mixthetworeagents1:1(~1mlperblot)andPipette~1mlonapieceofSaranwraptapedtothebench.
- Dragtheblotalongtheedgeofthetray,drainexcessTBST,thenplacetheblotwithproteinsidedownontheECLsolution.
- Incubatefor1’thendrainexcessreagentandtransfertheblottoaplasticreportcover.
- Exposeimmediately(fewseconds—1hr).
Stripping
- Afterdetectionwashthemembrane2x10’inTBST.
- Incubate30’at50degCinaclosedcontainerinstrippingbuffer(65mMTris-HClpH6.7,100mMbeta-mercaptoethanol,2%SDS).Ifstrippingisincompleteincreasetemperatureto60or70degC.
- Wash3x10’inalargevolumeofTBSTatRT.
- Themembraneisnowreadytobeblocked.
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