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Yeast Protocols Handbook
CLONTECH Laboratories, Inc.
IV. Preparation of Yeast Protein Extracts
A. General Information
We provide two alternative protocols for the preparation of protein extracts from yeast. The results
(i.e., protein yield and quality) will vary depending on the protein and may be more successful with
one protocol than with the other. Because it is difficult to predict which procedure will give better
results, we provide two protocols for comparison. The cell culture preparation method (Section B)
is the same for both protein extraction procedures.
Both extraction procedures address the two most challenging aspects of isolating proteins from
yeast: 1) disrupting yeast cell walls; and 2) inhibiting the many endogenous yeast proteases. Yeast
cell walls are tough and must be disrupted by a combination of physical and chemical means;
methods that utilize glycolytic enzymes are not recommended for this application because they are
often contaminated with proteases. Endogenous proteases must be counteracted with a cocktail
of strong protease inhibitors (recipe in Appendix D.A). If you know your protein of interest is
susceptible to a protease not inhibited by the recommended cocktail, add the appropriate inhibitor
before using the mixture. You may also wish to add other inhibitors such as sodium fluoride to
prevent dephosphorylation, if that is appropriate for your protein.
B. Preparation of Yeast Cultures for Protein Extraction
Reagents and Materials Required:
• YPD and appropriate SD liquid medium (Recipes in Appendix C.A)
• 20- and 50-ml culture tubes
• Ice-cold H2O
• Dry ice or liquid nitrogen
1. For each transformed yeast strain you wish to assay in a Western blot, prepare a 5-ml overnight
culture in SD selection medium as described in Section III.A, except use a single isolated
colony (1–2 mm in diameter, no older than 4 days). Use the SD medium appropriate for your
system and plasmids (Appendix E). Also prepare a 10-ml culture of an untransformed yeast
colony in YPD or (if possible) appropriate SD medium as a negative control.
2. Vortex the overnight cultures for 0.5–1 min to disperse cell clumps. For each clone to be
assayed (and the negative control), separately inoculate 50-ml aliquots of YPD medium with
the entire overnight culture.
3. Incubate at 30°C with shaking (220–250 rpm) until the OD600 reaches 0.4–0.6. (Depending on
the fusion protein, this will take 4–8 hr.) Multiply the OD600 (of a 1-ml sample) by the culture
volume (i.e., 55 ml) to obtain the total number of OD600 units; this number will be used in
Sections C & D. (For example, 0.6 x 55 ml = 33 total OD600 units.)
Note: During late log phase the ADH1 promoter shuts down and the level of endogenous yeast proteases increases.
4. Quickly chill the culture by pouring it into a prechilled 100-ml centrifuge tube halfway filled with
ice.
5. Immediately place tube in a prechilled rotor and centrifuge at 1000 x g for 5 min at 4°C.
6. Pour off supernatant and resuspend the cell pellet in 50 ml of ice-cold H2O. (Any unmelted ice
pours off with the supernatant.)
7. Recover the pellet by centrifugation at 1,000 x g for 5 min at 4°C.
8. Immediately freeze the cell pellet by placing the tube on dry ice or in liquid nitrogen. Store cells
at –70°C until you are ready to proceed with the experiment.
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Protocol # PT3024-1
Version # PR7X265