2-D sample prep

Fri Jun 3 09:28:31 EST 1994

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 

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.

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!

I'm getting frantic, I've been at this for 6 weeks, hopeful


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