quantification of protein

A.F. Simpson AFS7 at le.ac.uk
Wed Sep 22 12:11:59 EST 1999


AKB wrote:
> 
> I know the structure of a protein.  How do I quantify the amount of protein
> in a bacterial cell (or the lack thereof). 

> I need to determine if a protein's
> production is being inhibited by a specific antibiotic.  This is
> hypothetical and not from any specific experiment.

A western blot is a good way to start.

Have two samples - treated and untreated.  Load an equal amount of
_total protein_ onto the western.  Detect with antibody.  This will show
you if the expression of your protein is specifically inhibited beyond
any effects the antibody might have on total protein expression by the
cell.

I would recommend running a dilution series for the samples to make sure
you don't overload your protein and mask any reduction.

If you wish you can then quantify the bands via densitometry.  It's not
the most accurate technique in the world and won't be good enough to
detect small changes in expression, but it should be enough to let you
know if it's worth further investigation.

love
Anna



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