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人血管内皮细胞生长因子D(VEGFD)Elisa试剂盒_人Elisa试剂盒上海...

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MikeSchultz,PavelAprikian,SteveHahn

lastmodified6/28/99

Grow2litersyeastinYPD(with3%glucose)toanA600of1.5to2.0(inoculate~1.5mlsaturatedovernightin1litermediaandgrow~14hrat30degrees).Expect5-6gofcells.

Harvestcellsandwashcellsoncein30mlcoldH2O.

Washcellsoncein30mlcoldextractionbuffer.

Weighcellpellet.

Scrapepelletintoa10mlsyringeandsquirtintoa50mlscrewcaptubewhichissUSPendedintoliquidN2(thetubecanalsocontainalittleliquidN2ifdesired).Thiscellpelletcanbefrozenat-70deg.foratleastoneyearwithnolossofactivity.

Precoolalarge(~8inchdiameter)mortarandpestle.Putondryice.AddalittleliquidN2tocool.

Dumpinfrozenyeastandgrindbigpiecesuntileverythingisuniformlysmallpowder(~1min).

AddalittlemoreliquidN2beingcarefultonotmakepowderflyallover.

Grindagainforabout1min.RepeatliquidN2additionand1mingrinding.

Useaprecooledspatulatoscrapethepowderintoaprecooledweighboat.Dumpintoa50mlconicaltube.Storetubeondryiceifgrindingotheryeastcells.

Afterallgrindingiscomplete,transfertubetoiceandadd15%thetotalvolumeofextractionbuffer(i.e5gcells+750ulbuffer)andletsitonice20min.ScrapetheyeastsorbetintoanultraclearSW55tubeandspininultracentrifuge100,000xgfor1hrat4deg.(33,000rpminSW55).

Afterspinning,~1/3volumeiscellpellet,~2/3volumeaclearyellowliquidandathinwhitelipidlayeronisontop.Removeclearliquidwithasyringebeingcarefultoavoidthepelletandlipidlayer.

Dialyze3X1hrat4degreesvs500mldialysisbuffer.

Aliquotandfreezeextracts.

Expect20-30mg/mlprotein.Typically,use20microgramsinaPolIIIinvitrotranscriptionreaction.

SolutionsneededforWCE:

ExtractionBuffer(500ml)

100mMHEPES,7.9	11.9gHEPES
245mMKCl	9.14gKCl
5mMEGTA	10ml0.25MEGTA
1mMEDTA	2ml0.25MEDTA
	H20to500ml

Justbeforeuse,addproteaseinhibitorsandaddDTTto2mM.

DialysisBuffer1.5liters:

20mMHEPES,7.9	7.14gHEPES
100mMKCl	11.2gKCl
5mMMgCl2	7.5ml1MMgCl2
1mMEDTA	6ml0.25MEDTA
20%glycerol	300mlglycerol
	H20to1.5liter

Justbeforeuse,addproteaseinhibitorsandaddDTTto2mM.

ProteaseinhibitorsandDTT:

0.1MPMSF(100x)

16mg/mlEthanol

Storeat-20degrees

0.2MDTT

32mg/mlH2O

Storefrozenat-20degrees

Benzamidine(100X)

31mg/mlH2O.

Storefrozenat-20degrees

Leupeptin(500X)

0.15mg/mlEthanol.

Storeat-70degreesforlessthan6months

Pepstatin(200X)

0.28mg/mlmethanol

Storeat-20degrees.

Chymostatin(2,500X)

5mg/mlDMSO

Storefrozenat-20degrees

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