PreparationofYeastProteinExtracts

(seeClontechYPH,p12)

Crackingbufferstocksolution(seep54ClontechYPH)

8MUrea;5%w/vSDS;40mMTris-HClpH6.8;0.1mMEDTA;0.4mg/mlBMBinH2O

forafinalvolumeof20ml9.6gUrea

1gSDS

0.8ml1MTrisHClpH6.8

4µl0.5MEDTA

8mgBromophenolBlue

Protocol

1.Setup5mlO/NculturesincorrectSDselectionmedium(SD-LeuorSD-Trp).

2.Alsosetupuntransformedyeastasacontrol.Leaveat30°C,230-250rpm,for18-24h.

3.Foreachclonetobeassayed,separatelyinoculate50mlofYPDAwiththeentireO/Nculture.TakeanOD600reADIng.

4.Incubateat30°C,230-250rpm,untiltheOD600reaches0.4-0.6(thiscantakebetween4-8h).

5.UseOD600Unitstodeterminethevolumeofeachyeastculturetobetaken;i.e.decidetotake15OD600Unitsofeachculture15/OD600=volumetobetaken,soifOD600=0.5,take30ml

6.Foreachculture,transferthevolumecalculatedaboveintoa50mlFalconhalffilledwithice.Spinat2500rpm(MSE)for5min.

7.PouroffsupernatantandresUSPendpelletinto50mloficecoldH2Oandspinagain.

8.RemovesupernatantandfreezethepelletinliquidN2(cellscanbestoredat-80°C)

9.Justbeforeuse,makeupcompletecrackingbuffer

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