Quick and Easy Yeast Extract for Western Blots

1.     Grow yeast in a 5mL culture overnight to saturation.

2.     Measure the OD­₆₀₀ of the cultures.  If using a standard spectrophotometer, dilute the culture 1:10 in media and measure the OD of the diluted culture.  Calculate the OD of the culture by multiplying by the dilution factor (e.g. 10).

3.     Spin down 2.5OD₆₀₀ units of culture at 10K rpm for 2 minutes. An OD unit = mL x OD₆₀₀ of cells (e.g. 2.5 OD unit = 1 ml at OD₆₀₀ 2.5. )

                  Sample Calculation:

                  Yeast culture OD₆₀₀= 3.5

                  2.5 OD unit = 2.5/3.5 = 0.714 mL of culture

4.     Remove supernatant and resuspend in 100uL of water.

5.     Add 100uL 0.2M NaOH and mix well.  Incubate at room temperature, 5 minutes.

6.      Pellet cells by spinning at 10Krpm for 2 minutes. 

7.     Remove supernatant and resuspend pellet in 50uL 1x SDS loading buffer. Boil for 3 minutes. 

8.     Pellet debris by spinning at 10Krpm for 1 minute.  Load 6uL of supernatant on to an SDS-PAGE gel.

Required Solutions:

0.2M NaOH

0.4 g NaOH in 50mL H₂0

5X SDS loading buffer

250mM Tris-HCl pH 6.8

10%SDS

30% glycerol

5% ß-mercaptroethanol

0.02% bromphenol blue