A Dohlman Lab Protocol
Whole Cell Extracts
REFERENCE: Hoffman, G., Garrison, T. R., and Dohlman, H. G., Analysis of RGS proteins in Saccharomyces cerevisiae, Methods Enzymol.344:617-631, 2002.
-Use sterile technique and sterile solutions in steps 1 to 3.-
1. Using a saturated starter culture, inoculate 25 to 30 ml of appropriate media in a 125 ml flask.
***Since it is often difficult to estimate the growth rate of yeast, it is helpful to start several 25 ml cultures, each with a different dilution of the starter culture (e.g. 1:100, 1:300, 1:900).
2. Grow at 30 C shaking (250 rpm) until the OD600 nm ~ 1.0 (This is usually done overnight).
***When growing several strains at once, it is likely that they will all reach OD600 nm ~ 1.0 at different times. If desired, sodium azide (1M stock in water, diluted to a final concentration of 10 mM) can be added to a culture once it reaches an OD600 nm ~ 1.0. The culture can then be placed on ice until the others are ready.
3. Transfer to a 50 ml conical tube and centrifuge for 10 min at 2000 x g at 4 C.
4. Resuspend each sample in 1 ml of 10 mM sodium azide and place on ice.
5. Calculate the volume of resuspended cells that would translate to an OD600 nm reading of 10. For example, this would equal 1 ml if 10 ml of culture at OD600 nm = 1.0 had been centrifuged and resuspended.
***This step is necessary to equalize the amount of cells (and protein) in a given volume of whole cell extract.
6. Transfer the calculated volume of resuspended cells to a microfuge tube and centrifuge at 16,000 x g for 1 min.
7. Aspirate the supernatent.
8. Resuspend the pellet in 200 ul of 1X SDS-PAGE sample buffer.
9. Immediately place in a 100 C heat block for 10 min.
10. Allow the tube to cool and add 200 ul of glass beads (Sigma, #G-8772).
11. Vortex at high speed for 2 min. Invert after the first min.
***Several tubes can be vortexed at the same time by using a foam tube floater to hold them together.
12. Using a 21 gauge needle, poke a hole in the bottom of each tube and place it into a new microfuge tube.
13. Centrifuge at 2000 x g for 10 sec to expel the liquid into the bottom tube, leaving the glass beads in the top tube.
14. Discard the glass beads and centrifuge the bottom tube at 16,000 x g for 2 min. This sediments any insoluble material.
15. Transfer the supernatant to a new microfuge tube. Store at -20 C.
16. When ready to use, heat at 37 C for 10 min, vortex, and centrifuge at 16,000 x g for 1 min.
***Keep in mind that repeated freezing and thawing can degrade the protein sample.
17. Immunoblots can be performed using standard methods.
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