Cloning of GST-E2F-1: HELP!

[Andrew Ippolito]

I am attempting to isolate various mutants of the E2F protein
grown up in pGEX in e.coli.  I have attempted this using
our standard protocol... 2 ml of overnight culture into 200 ml
LB, grown to an OD(600) of ~.5, addition of 1 mM IPTG, induced
for 3.5 hours, spun down, sonicated and freeze-thawed, washed
in NETN followed by Elution buffer, followed by elution buffer
with 30.7 mg glut. per 5 ml E.B.  
this is a rough outline.  
I did not get much of anything when i stained a SDS PAGE of the
samples.  Help! please send me a personal e-mail, as I do
not frequent this newsgroup very often.  Thank you in
advance,
Andrew


Andrew Ippolito
Dept. Molecular & Cellular Biology
Roswell Park Cancer Institute
ippo at scientist.com
mr-drew at yahoo.com

"I know not what weapons World War III will be fought
with, but I know that World War IV will be fought
with sticks and stones."
-Albert Einstein