Protein extraction from yeast1. Glass beads lysisConzelmann A, Riezman H, Desponds C, BronC.1988. A major 125 kd membraneglycoprotein of Saccharomyces cerevisiae is attached to the lipid bilayer through aninositol-containg phospholipid. EMBO J 7: 2233±2240.2. rapid protein extraction in optimized SDS sample bufferHorwathA,RiezmanH.1994.RapidproteinextractionfromSaccharomycescerevisiae. Yeast 10: 1305±1310.Sample Buffer:0.06M Tris-HCl, pH6.810% (v/v) glycerol2% (w/v) SDS5% (v/v) 2-mercaptoethanol0.0025% (w/v) bromophenolblue10 ml0.6 ml 1M Tris6.82 ml 50% glycerol2 ml 10% SDS0.5 ml 2-mercaptoethanol0.1 ml Sat. Bromphenolblue4.9 ml H2OMake sample Buffer fresh before use. Can store buffer frozen at—20 degrees for ~6 months.1. Grow cells overnight (~1x107cells/ml; A600 =0.7) and collect1.5 ml cells (adjustvolumes according to cell density of cultures) in1.5 ml microfuge tube (1 minute, 14000xg).It is important not to grow the cells to a high density.10 microliters of a saturated overnight culture in YPD innoculated to 5 ml SD + essentialamino acids for ~16 hrs gives A600 of0.5 to1.0 for wild-type cells grown at 30 degrees150 microliters of YPD saturated culture diluted to 5 ml YPD and grown for ~5 hrs at 30degrees gives an A600 of ~0.8 for wild-type cells.2. Wash cells 1X with water and collect again by centrifugation.3. Resuspend cells in 100 microliters sample buffer.4. Heat at 95 deg C for 5 minutes.5. Centrifuge 14000xg for 5 minutes. Load 15 microliters per lane on an SDS PAGE3. post-alkaline extractionVitalyV.Kushnirov2000.Rapidandreliableproteinextractionfromyeast.Yeast2000; 16: 857±860.about2.5 OD600 (which constitutes about2.3 mg of wet weight) of yeast cells wereharvested by centrifugation from liquid culture or scraped off the agar plate usingabacteriologicalloop.Thesecellsweresuspendedin100mldistilledwater,added100 ml0.2 M NaOH, incubated for 5 min at room temperature, pelleted, resuspendedin50mlSDSsamplebuffer,boiledfor3minandpelletedagain.About6μlsuper natantwastypicallyloadedperlaneofmini-gel(Bio-RadMini-Proteancell).Thesamplebuffer(0.06MTris±HCl,pH6.8,5%glycerol,2%SDS,4%bmercaptoethanol,0.0025% bromophenolblue) was slightly modi®ed fromstandard(Laemmli, 1970).SDS-Boil-Beads Whole Cell ExtractsREFERENCE:Hoffman,G.,Garrison,T.R.,andDohlman,H.G.,AnalysisofRGS proteins inSaccharomyces cerevisiae, Methods Enzymol.344:617-631,2002.-Use sterile technique and sterile solutions in steps 1 to3.-ingasaturatedstarterculture,inoculate25to30mlofappropriatemedia in a 125 ml flask.***Since it is often difficult to estimate the growth rate of yeast, itishelpfultostartseveral25mlcultures,eachwithadifferentdilutionof the starter culture (e.g. 1:100, 1:300, 1:900).2.Growat30Cshaking(250rpm)untiltheOD600nm~1.0(Thisusuallydone overnight).***When growingseveral strains atonce, itis likely thatthey willallreachOD600nm~1.0atdifferenttimes.Ifdesired,sodiumazide(1Mstockin water, diluted to a final concentration of 10 mM) can be added to acultureonceitreachesanOD600nm~1.0.Theculturecanthenbeplacedon ice until the others are ready.3. Transfer to a 50 ml conical tube and centrifuge for 10 min at 2000 xg at 4C.4. Resuspend each sample in 1 ml of 10 mM sodium azide and place on ice.5. Calculate the volume of resuspended cells that would translate to anOD600 nm reading of10. For example, this would equal 1 ml if 10 ml ofculture at OD600 nm =1.0 had been centrifuged and resuspended.***This step is necessary to equalize the amount of cells (and protein)in a given volume of whole cell extract.6.Transferthecalculatedvolumeofresuspendedcellstoamicrofugetubeand centrifuge at 16,000 x g for 1 min.7. Aspirate the supernatent.8. Resuspend the pellet in 200 ul of 1X SDS-PAGE sample buffer.9. Immediately place in a 100 C heat block for 10 min.10.Allowthetubetocoolandadd200ulofglassbeads(Sigma,#G-8772).11. Vortex at high speed for 2 min. Invert after the first min.***Several tubes can be vortexed at the same time by using a foam tubefloater to hold them together.12. Using a 21 gauge needle, poke a hole in the bottom of each tube andplace it into a new microfuge tube.13.Centrifugeat2000xgfor10sectoexpeltheliquidintothebottomtube, leaving the glass beads in the top tube.14. Discard the glass beads and centrifuge the bottom tube at 16,000 xg for 2 min. This sediments any insoluble material.15. Transfer the supernatant to a new microfuge tube. Store at -20C.16. When ready to use, heat at 37 C for 10 min, vortex, and centrifugeat 16,000 x g for 1 min.***Keepinmindthatrepeatedfreezingandthawingcandegradetheproteinsample.17. Immunoblots can be performed using standard methods.Small Scale Yeast Whole Cell Extract for IPSteve HahnAugust 2007Grow 100 ml yeast cells in desired media overnight to an A600 of ~1.0 (0.6 to1.2 workswell).Forgrowthinminimalmedia,1mlofasaturatedovernightcultureinminimalmedia(Synthetic dextrose (SD) with only the required amino acids) was inoculated to 100 ml of thesame media and grown ~16 hrs at 30 degrees.Harvest cells and wash with 20 ml of cold extraction buffer in a 50 ml tube.Resuspendcellsin0.5mlextractionbuffercontaingDTTandproteaseinhibitorsinamicrocentrifuge tube with a locking top (marsh tube).Add ~500 microliters of glass beads. In the cold room, shake tubes on the foam ring of thevortex mixer platform at top speed for 1 min. Transfer to ice for 1 min. I have done up to 20extracts at once.Repeat for a total of 10 min of vortexing.Briefly microcentrifuge to remove all liquid and leave behind most of the glass beads.Centrifuge at top speed at 4 degrees for 15 min and remove supernatant, being careful toavoid any glass beads.Assay protein concentration using BioRad or Pierce assays. Freeze extracts and store at -80deg. Expect 10-15 mg/ml protein.Extract Buffer:100 mM Tris pH7.9250 mM Ammonium Sulfate1 mM EDTA10% GlycerolBefore use, add DTT to0.5 mM (low concentration so IP reactions can be done directly)And 1X protease inhibitors from the following stock solutions:0.1 M PMSF (100x) 16 mg/ml Ethanol; Store at -20 degreesBenzamidine (100X); 31 mg/ml H2O; Store frozen at -20 degreesLeupeptin (500X);0.15 mg/ml Ethanol; Store at -70 degrees for less than 6 monthsPepstatin (200X);0.28 mg/ml methanol; Store at -20 degrees.Chymostatin (2,500X); 5mg/ml DMSO; Store frozen at -20 degreesTCA protein precipitationTo concentrate proteins for analysis by SDS PAGE:If a small amount of protein is to be precipitated (less than a few micrograms), add Insulin asa carrier protein (10 micrograms of Sigma insulin, I-5500, per sample works well).1.Add an equal volume of 20% TCA (trichloroacetic acid) to protein sample.2.Incubate 30 min on ice.3.Spin in microfuge at 4 deg. For 15 min.4.Carefully remove all supernatant.5.Add ~300 ul cold acetone and spin 5 min at 4 degrees.6.Remove supernatant and dry pellet.7.Resuspend samples in SDS PAGE loading buffer. Load to SDS PAGE after heating at65 deg for 3 min.Acetone precipitation of protein1. Cool the required volume of acetone to -20°C.3. Add six times the sample volume of cold (-20°C) acetone to the tube.4. Vortex tube and incubate for 2 hours to overnight minutes at -20°C.5. Centrifuge 15 minutes at 13,000-15,000 x g at 4°C.6. Decant and properly dispose of the supernatant, being careful to not dislodge the proteinpellet.7. Briefly wash the pellet with 100ul of cold 90% acetone.8. Centrifuge 5 minutes at 13,000-15,000 x g at 4°C.9. Remove sup and repeat if necessary.10. Air dry for ~15-30 minutes and resuspend in an appropriate buffer.。