PhastSystem/Western

John H Richburg John_H_Richburg at Brown.edu
Thu Feb 15 16:27:04 EST 1996


I am having trouble getting good protein separation using the PhastSytem
so that I may ultimately do western blotting.

I suspect my trouble is in the sample preparation. 

I homogenized my tissue with Dounce homogenizer in buffer containing 0.1 g
CHES, 0.2 g SDS, o.1 g DTT, 1 ml glycerol in a volume of 10 ml (1.5 ml/100
mg tissue). 
I then centrifuged for 2 h at 100,000 x g.
My final protein conc. is 1 mg/ml.

To run on the gel. I did a TCA ppt and solubilized proteins in 0.1 N NaOH.
I added 7.5 µl of sample to 2.5 ml sample buffer (50 mM Tris, 10%
glycerol, 2% SDS, 0.1% BPB + mercaptoethanol).

I boiled. Then placed 4 µl of sample (=3 µg protein) on 20% homogeneous
gel for separation using SDS buffer strips.

Coomasie stianed gel reveals a smear that is wide at the origin leading to
a point at the bottom of the gel.

Could an experienced protein chemist/Phast user shed insight into my messy
separation. Western blot of this gel gave only two non-specific bands near
the origin.
My proteins of interest are 26 and 40 Kd.

Help!

John

John_H_Richburg at brown.edu

-- 
John Richburg
Dept. Pathology and Laboratory Medicine
Brown University

John_H_Richburg at brown.edu



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