streaky page gels

[Andrew J. Cary]

I'll throw in my two cents.  I have had streaky gels twice.  Once when
there was not enough SDS in the running buffer.  The sds was slowly
titered off during the run and so made a nice smear down the lane.  This
is probably not your problem since your standards run fine.  The other was
when I had too much detergent in my sample (I am working with a membrane
receptor).  This caused streaking down the whole lane.  You might be
carring over something even through the precipitation etc.  Maybe changing
your clean up would help.  Perhaps a de-salting gel filtration column
prior to the TCA precipitation???  

Good Luck
Andy



> > I am still having trouble so let me give you alittle more information
> > about my problem.  I have done a couple rounds of IE chromatography on
> > Xenopus egg extracts and am running the fractions that were positive by
> > western over a Superose6 size exclusion column.  My protein is quite large
> > so I am running a straight 6%gel with no stacker as my protein seems to
> > get hung up at the interface when I include a stacker.  After the SE
> > chromatography, I am TCA precipitaing the fractions and resuspending in a
> > standard 1X loading buffer.  The prestained markers look fine and by
> > looking at the dye front, I don't see anything weird about how the gel
> > is running.  I am blotting with the same Ab that I used to verify the
> > presence of my protein in previous steps with  the same conditions.
> > However, when I run these samples, all I see after developing my western
> > are linear streaks running straight up and down where my lanes are and no
> > detectable bands.  Thanks to everyone who has written so far, I am
> > completely flumoxed by this, and am ready to throw the column out.
> >
> > T. Patrick Purk

-- 
Andrew J. Cary
Dept of Molecular, Cellular and Developmental Biology 
Yale University