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

Protein extraction from yeast 1. Glass beads lysis Conzelmann 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: 2233±2240. 2. rapid protein extraction in optimized SDS sample buffer Horwath A, Riezman H. 1994. Rapid protein extraction from Saccharomyces cerevisiae. Yeast 10: 1305±1310. Sample Buffer: 10 ml 0.06M Tris-HCl, pH 6.8 ? 0.6 ml 1M Tris 6.8 10% (v/v) glycerol ? 2 ml 50% glycerol 2% (w/v) SDS ? 2 ml 10% SDS 5% (v/v) 2-mercaptoethanol ? 0.5 ml 2-mercaptoethanol 0.0025% (w/v) bromophenol blue ? 0.1 ml Sat. Bromphenol blue ? ? 4.9 ml H2O Make 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 degrees 150 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 PAGE 3. post-alkaline extraction Vitaly V. Kushnirov 2000. Rapid and reliable protein extraction from yeast. Yeast 2000; 16: 857±860. 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 m