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unstable protein expression - (Apr/17/2009 )

Hi,

Hope I can get some help with a problem I am having with inconsistent protein expression.
I am using whole cell lysate for a western blot, but am getting inconsistencies in protein expression.
I seed on a 6 well plate, apply proteasome inhibitor at different concentrations (and on a time course scale) and compare with a negative control.

The protein is expressed on all samples... the negative control has degradation products (bands smaller than the whole protein), while the MG132 treated samples have the target protein, no degradation products, and ubiquitynated products... ideal!!

The second and third time I performed the experiment however, I get no degradation products and basically no ubiquitynation. All lanes look the same.
What exactly am I doing wrong?

Cells were seeded at equal numbers in all cases, and there is basically no difference in the way I go about doing the experiment.

Am thinking about applying TCA before lysis.

Any help is very much appreciated.

-kokoro-

What expression system did you use? E coli? Yeast? Insect? etc?
How did you extract your protein? Sonication? Bead? French Press? etc?
What buffer too? etc
etc...
etc...

-genz-