2-D sample prep

Mark J. Miller mark at alw.nih.gov
Fri Jun 3 13:57:58 EST 1994

In article <2snemf$77g at mserv1.dl.ac.uk>, GAED6043 at VAX1.AGRICULTURE.QUEENS-BELFAST.AC.UK writes:
|> Mega problem!
|> I'm running 2-D gels (Bio-rad protean II) of total E.coli protein and have several problems:
|> 1.   IEF,  according to Bio-Rad when the voltage is set to 200V the current running should be
|> 3.5mA -  all I can manage is 1mA.  (4 of 14cm x 1.5mm tube gels 
|> per side)  The electrolytes are 20mM NaOH and 10mM H3PO4 
Remember that the IEF tubes are set up "in parallel". Hence the more tubes, the higher the initial
current. We run at 25 mW/tube, constant power untill we reach 800V, then switch to constant volts.
Initially, we get from 100-150 microamps/gel, so your 1 ma for four gels is high, but not
unreasonable. As the pI gradent is set up, the current drops dramatically: 5-10X. If it doesn't,
your gradient is not being set up. Most common reason: electrolytes reversed (i.e. acid in the
upper chamber, not lower -- we all do it at some time).

|> 2.  Although the current is not what it's supposed to be the molecular weight markers run
|> but the bands are broad - as seen in the second dimension.
Try using the BioRad colored IEF markers. They'll show you if the samples are focusing or not.

|> 3.  When I run E.coli protein I get nothing on the second dimension gel - amount loaded onto
|> the first dimension is approx. 500ug total protein!
Are your proteins properly solubilized? E.coli can be a tough bug to bust. Is the sample buffer
9M urea, have you centrifuged the sample prior to loading? 

|> I'm getting frantic, I've been at this for 6 weeks, hopeful
|> kim
If this fails, remake all your solutions from scratch. Double check all calculations. Make sure
you haven't substituted a milli for a micro. These kinds of mistakes can be the hardest to
catch because "I haven't done anything that dumb since high school!" Good luck.

Mark J. Miller                  phone: (301) 496-9343 or 496-5688
Building 37, Room 3C28          fax:   (301) 402-2867 or 496-0734
National Institutes of Health   Internet: mark at helix.nih.gov
Bethesda, MD 20892

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