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Preparation of Yeast Cell Lysate for Western Blotting

Posted on Wednesday, October 29, 2003

Description
This method is a way of quickly making a crude cell lysate from your yeast cells which is ready to run on a western blot, without any need for bead bashing or enzymatic treatment.

Procedure
1. Spin down 20ml overnight yeast culture and wash once using distilled water.
2. Suspend pellet in 0.5-1ml loading buffer and transfer cells to a locking microcentrifuge tube.
3. Place tube in a 100 degrees C hot block and boil sample for 5min.
4. Remove tubes to ice for 5min to chill.
5. Spin tubes for 5min at max speed in a microcetrifuge (preferably chilled).
6. Remove supernatant to a fresh tube. This supernatant is now ready for protein determination (by Lowry method) and direct loading onto protein gel.
7. Unused samples can be stored, frozen in this buffer.

Recipes
Loading Buffer:
1.2ml 1M Tris (pH6.8)
2ml glycerol
4ml 10% SDS
1ml Mercaptoethanol
0.1ml protease inhibitor cocktail for yeast (Sigma P8215)
1.7ml water

Supplies
1.5 or 2ml Boiling tubes
Reagents to do Lowry protein assay

Tips
It is important not to use the Braqdford method of protein determination with these extracts because the SDS in the loading buffer will interfere with the assay. It is better to use the Lowry method (or something similar). Biorad sell the reagents to do this under the name of "DC Protein Assay"

Submitted by: Gillian

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