Rapid Yeast Protein Prep for SDS PAGE and Western

2024-11-25 蛋白质技术加入收藏

Sample Buffer:10 ml0.06M Tris-HCl, pH 6.80.6 ml 1M Tris 6.810% (v/v) glycerol2 m

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.

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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 as this method will not work well.

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 polyacrylamide gel.

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