Flaws in this protocol? (TCA-extraction-yeast)

zaxxon zhowar1 at umbc.edu
Fri Apr 3 13:19:15 EST 1998


Could anyone suggest where in this protocol I might be losing protein?

It works well occasionally but I've never been able to get it to work
consistently over a period of time. l seem to be losing protein
somewhere. Sometimes in one or two samples, sometimes all of them. I need
equal harvesting of about 12 samples in order to compare the amount of a
particular protein over time.

With Sacchormyces cerevisiae:

1. Harvest 800 ul aliquots (always at same cell density)
2. Add 160 ul (1.85 M NaOH, 7.4% Beta-mercaptoethanol)
3. 20' on ice
4  Spin at 4C 10'
5. Add 100 ul 100% TCA (final conc. ~10%)
6. 20' on ice
7. Spin at 4C 10'

Repeat twice:
1. Remove all supernatant with pipet tips. Add 1 ml acetone. Vortex.
2. Sit 30' on ice.
3. Spin 10' at 4C

Then:
1. Remove final acetone supernatant as well as possible manually.
2. Air dry 10' under hood
3. Add 50 ul 1x buffer (4% SDS, etc) 
4. Manually resuspend pellet with pipet tip.
5. Seperation by SDS-PAGE


Any ideas would be welcome.

I've thought perhaps:
1. I'm not cracking open the cells well
2. I'm not precipitating efficiently with the TCA (Can NaOH interfere?)
3. I'm physically losing protein when I remove supernatants, perhaps by
disturbing the pellet.  
4. The protein is not solubilizing in my loading buffer. Could be
interference by residual TCA, or acetone. Can acetone interfere with
solubilization? 

As far as not removing TCA: my samples used to turn yellow (acidic)
occasionally so I thought I wasn't removing the TCA well. I increased the
amount and time in acetone and for a while I seemed to get a lot better
extraction. Lately it's gotten more variable again. 



Thanks,

Zach




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