Protein Extraction for Yeast

This was my main protocol for preparing samples for SDS-Page gels and western blots.

MURBs Buffer Recipe

1. 50mM sodium phosphate
2. 25mM MES
3. 3M urea
4. 0.5% 2-mercaptoethanol
5. 1 mM sodium azide
6. 1% SDS
7. Blue color dye

Protocol

1. Grow yeast cells until they are in log-phase.
2. Collect 10-15ml of samples. It is possible to do this with 1ml of sample as long as the pellet is large enough.
3. Centrifuge at 3000rpm for 2-3 minutes and aspirate.
4. Resuspend in 1ml of 10% TCA, move samples to a 1.7ml microcentrifuge tube, and sit on ice for 20 minutes.
5. Spin down and aspirate TCA. You can snap freeze here and store at -20C or continue with the extraction.
6. Wash with acetone twice and allow the pellet to air dry. This is important as any leftover TCA will cause your proteins to precipitate out during the extraction.
7. Add MURBs buffer. For 10-15ml samples I will add 200-500ul of buffer. You can adjust based on pellet size.
8. Add acid washed beads and shake for 2 minutes to lyse the cells. If the buffer turns yellow you can add more of the MURBs buffer until it turns blue.
9. Poke a hole in the bottom of the 1.7ml tube and place in a 15ml tube.
10. Spin down and collect the supernatant.
11. Boil for 10 minutes.

Reference

1. Miller-Fleming, L., et al.,Detection of Saccharomyces cerevisiaeAtg13 by western blot. Autophagy, 2014. 10(3): p. 514-7.