Protein extraction from yeast(酵母蛋白质提取).doc

Protein extraction from yeast1. Glass beads lysisConzelmann A, Riezman H, Desponds C, Bron C. 1988. A major 125 kd membrane glycoprotein of Saccharomyces cerevisiae is attached to the lipid bilayer through an inositol-containing phospholipid. EMBO J 7: 22332240.2. rapid protein extraction in optimized SDS sample bufferHorwath A, Riezman H. 1994. Rapid protein extraction from Saccharomyces cerevisiae. Yeast 10: 13051310.Sample Buffer: 10 ml0.06M Tris-HCl, pH 6.80.6 ml 1M Tris 6.810% (v/v) glycerol2 ml 50% glycerol2% (w/v) SDS2 ml 10% SDS5% (v/v) 2-mercaptoethanol0.5 ml 2-mercaptoethanol0.0025% (w/v) bromophenol blue0.1 ml Sat. Bromphenol blue4.9 ml H2OMake sample Buffer fresh before use. Can store buffer frozen at 20 degrees for 6 months.1. Grow cells overnight (1x107 cells/ml; A600 = 0.7) and collect 1.5 ml cells (adjust volumes according to cell density of cultures) in 1.5 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 + essential amino acids for 16 hrs gives A600 of 0.5 to 1.0 for wild-type cells grown at 30 degrees150 microliters of YPD saturated culture diluted to 5 ml YPD and grown for 5 hrs at 30 degrees 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 extractionVitaly V. Kushnirov 2000. Rapid and reliable protein extraction from yeast. Yeast 2000; 16: 857860.about 2.5 OD600 (which constitutes about 2.3 mg of wet weight) of yeast cells were harvested by centrifugation from liquid culture or scraped off the agar plate using a bacteriological loop. These cells were resuspended in 100 ml distilled water, added 100 ml 0.2 M NaOH, incubated for 5 min at room temperature, pelleted, resuspended in 50 ml SDS sample buffer, boiled for 3 min and pelleted again. About 6 l supernatant was typically loaded per lane of mini-gel (Bio-Rad Mini-Protean cell). The sample buffer (0.06 M TrisHCl, pH 6.8, 5% glycerol, 2% SDS, 4% bmercaptoethanol,0.0025% bromophenol blue) was slightly modied from standard (Laemmli, 1970).SDS-Boil-Beads Whole Cell ExtractsREFERENCE: Hoffman, G., Garrison, T. R., and Dohlman, H. G., Analysis of RGS proteins in Saccharomyces cerevisiae, Methods Enzymol.344:617-631, 2002. -Use sterile technique and sterile solutions in steps 1 to 3.- 1. Using a saturated starter culture, inoculate 25 to 30 ml of appropriate media in a 125 ml flask. *Since it is often difficult to estimate the growth rate of yeast, it is helpful to start several 25 ml cultures, each with a different dilution of the starter culture (e.g. 1:100, 1:300, 1:900). 2. Grow at 30 C shaking (250 rpm) until the OD600 nm 1.0 (This is usually done overnight). *When growing several strains at once, it is likely that they will all reach OD600 nm 1.0 at different times. If desired, sodium azide (1M stock in water, diluted to a final concentration of 10 mM) can be added to a culture once it reaches an OD600 nm 1.0. The culture can then be placed on ice until the others are ready. 3. Transfer to a 50 ml conical tube and centrifuge for 10 min at 2000 x g at 4 C. 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 an OD600 nm reading of 10. For example, this would equal 1 ml if 10 ml of culture 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. Transfer the calculated volume of resuspended cells to a microfuge tube and 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. Allow the tube to cool and add 200 ul of glass beads (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 tube floater to hold them together. 12. Using a 21 gauge needle, poke a hole in the bottom of each tube and place it into a new microfuge tube. 13. Centrifuge at 2000 x g for 10 sec to expel the liquid into the bottom tube, leaving the glass beads in the top tube. 14. Discard the glass beads and centrifuge the bottom tube at 16,000 x g for 2 min. This sediments any insoluble material. 15. Transfer the supernatant to a new microfuge tube. Store at -20 C. 16. When ready to use, heat at 37 C for 10 min, vortex, and centrifuge at 16,000 x g for 1 min. *Keep in mind that repeated freezing and thawing can degrade the protein sample. 17. Immunoblots can be performed using standard methods. Small Scale Yeast Whole Cell Extract for IPSteve Hahn August 2007Grow 100 ml yeast cells in desired media overnight to an A600 of 1.0 (0.6 to 1.2 works well). For growth in minimal media, 1 ml of a saturated overnight culture in minimal media (Synthetic dextrose (SD) with only the required amino acids) was inoculated to 100 ml of the same media and grown 16 hrs at 30 degrees.Harvest cells and wash with 20 ml of cold extraction buffer in a 50 ml tube.Resuspend cells in 0.5 ml extraction buffer containing DTT and protease inhibitors in a microcentrifuge tube with a locking top (marsh tube).Add 500 microliters of glass beads. In the cold room, shake tubes on the foam ring of the vortex mixer platform at top speed for 1 min. Transfer to ice for 1 min. I have done up to 20 extracts 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 to avoid any glass beads. Assay protein concentration using BioRad or Pierce assays. Freeze extracts and store at -80 deg. Expect 10-15 mg/ml protein.Extract Buffer:100 mM Tris pH 7.9250 mM Ammonium Sulfate1 mM EDTA10% GlycerolBefore use, add DTT to 0.5 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 precipitation To concentrate proteins for analysis by SDS PAGE:If a small amount of protein is to be precipitated (less than a few micrograms), add Insulin as a 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 at 65 deg for 3 min. Acetone precipitation of proteinThis procedure is suitable for recovering proteins from most aqueous solvents and from SDS c