2D gel HELP !

mary beard mbeard at oavax.csuchico.edu
Wed Mar 16 18:11:54 EST 1994




HELP!  I am a graduate student having a hell of a time with two-dimensional
electrophoresis of plant plasma membranes.  I isolate the membranes by
Larsson's aqueous two-phase method, resulting in a membrane pellet of about
1mg/ml protein as determined by BCA.  I use the method described by
Sandstrom et al to inhibit ATPase activity with vanadate.  Then I get
stuck, anyone have ideas on the following?:

Using Hurkman & Tanaka's method of phenol extraction of the membrane
proteins, I get precipitate in both phases.  This (protein) is difficult to
dissolve in a urea lysis buffer and even after clearing the sample I ALWAYS
get a precipitate/line across the top of the IEF gel (BioRad tubes).  The
second dimension gel show a long streak as if the sample sat on top of the
gel and didn't migrate.  SOMETIMES there are a few protein spots, but
nothing consistent.  I use IEF markers and get a color gradient down the
tube, I have had a successful second dimension gel of Sigma seven markers,
and I think I have seen ampholyte spots with silver stain, so I think the
problem is my sample or treatment/contamination of it.

Next, I tried Hurkman's method of 4% SDS buffer followed by acetone
precipitation, this gave the same result of a faint line across the IEF gel
and a streak on the second dimension.  At this point I needed to know both
dimensions were electrophoresing, and tried acetone precipitation of
supernate fractions only to end in the same brown streak!

1)  What is this line that forms across the IEF gel?  I overlay it with
urea as it polymerizes, prefocus the tubes, the top of the gel is never in
contact with air.  I have tried gradually increasing the voltage vs. high
voltage from the start.  Could prefocusing be changing the gel surface?

2)  What other methods are there to solubilize integral proteins?  I have
tried one dimension separation using NP-40 and Chaps (separately), but I
had a hard time removing the detergent (streaks).  I can't find a
reference, is there a problem using TCA with SDS?

3)  All the above have been tried MANY times, I feel confident my reagents
are pure and prepared right.

4)  Is there a reason I haven't seen many recent publications on two-
dimensional electrophoresis of plant membranes?  Should I convince my
advisor this brown streak is a sign of my frustration and that gradient
gels are going to be the extent of my thesis?


Thanks in Advance.
**********************************************************
MARY BEARD, CALIFORNIA STATE UNIVERSITY, CHICO
MBEARD at OAVAX.CSUCHICO.EDU



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