Sample prep for IEF-PAGE?

Tom Anderson ucgatan at socrates-a.ucl.ac.uk
Fri Jan 23 14:07:20 EST 2004


On 23 Jan 2004, OBF wrote:

> I am planning to do some experiments to determine the phosphorylation
> state of a 15 kDa regulator in my bug (a Bacillus sp.). I am intending
> to use IEF-PAGE to study this. The pI of the regulator should change
> upon phosphorylation and so I should be able (in principle) to
> seperate the two form by IEF-PAGE and then I can detect the protein by
> Western blotting. I will be using Bio-Rad's Criterion system and their
> precast gels. My question now is how I should treat the protein
> samples prior to loading. Can I just extract total protein extracts
> (with beadbeating) and then I plan to resuspend an aliquot of the
> extract in loading buffer (50% glycerol) and start the
> electrophoresis. Or should I first pretreat my sample to get rid of
> membrane/DNA/salts etc?

I'd guess you'd be okay with that; there is of course only one way to find
out, and that's to try it!

When i did IEF for proteomics, we would do a TCA precipitation to desalt
and a couple of acetone washes to delipidate; my samples were platelets. A
guy who was working on skin samples used to do six (i think) acetone
washes, as that's a rather fatty tissue. However, we did that because we
were aiming for the best possible resolution, as we were trying to map the
whole proteome; i think you'd be fine with some slight blurring. So,
unless your bugs are particularly fatty, or your lysis procedure is
particularly salty, i'd guess you can just resuspend.

Also, since your protein is only 15 kDa, you'd risk quite substantial
losses in any washes.

I don't know what Bio-Rad's tech support is like, but they might be the
best people to ask.

Hope this helps,
tom

-- 
Tom Anderson, MRC Laboratory for Molecular Cell Biology, UCL




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