Please leave your comments or questions

Quick and Easy Yeast Extract for Western Blots

Author: Erin M Green, updated date: , view: 269, Q&A: 0


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


30% glycerol

5% ß-mercaptroethanol

0.02% bromphenol blue



Please login to post your questions/comments. Your questions will be directed to the authors of the protocol. The authors will be requested to answer your questions at their earliest convenience. Once your questions are answered, you will be informed using the email address that you register with bio-protocol.
You are highly recommended to post your data including images for the troubleshooting.
You are highly recommended to post your data (images or even videos) for the troubleshooting. For uploading videos, you may need a Google account because Bio-protocol uses YouTube to host videos.