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



More information about the Methods mailing list