Problem: yeast protein remaining insoluble

Zach Howard zhowar1 at umbc.edu
Thu Sep 17 15:33:21 EST 1998


I'm working with an HA-tagged protein (OPI1) in S.cerevisiae

It's a putative transcription factor. No transmemebrane regions, but
potential myristoylation sites, so it may be membrane associated.

Here's the problem:

I've noticed that I get a lot more (after SDS-page, transfer,
immunoprobing,  ECL) of my protein if I load the insoluble
fraction with the soluble fraction. A lot more than if I just load the
soluble fraction.  This is following TCA extraction with 2-3 acetone or
ethanol washes.  The problem is if I load the insoluble fraction I get
really ugly bands.  

Has anybody encountered this? Obviously my protein is remaining in the
insoluble fraction. 

Could this be due to failure to remove all of the TCA, or possibly the
protein is associating with an insoluble cell component?

This occurs whether or not I heat the sample.

Thanks,

Zach





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