NEED HELP with non-denaturing IEF

Aye M.Tin muamt2 at
Mon Sep 25 14:59:47 EST 1995


I am a student trying to visualize peroxidase isozyme bands using IEF 
under non-denaturing conditions. I am using 5% acrylamide, 10% glycerol, 
and 3% ampholytes (3-10) for my slab gel. (vertical slab gel). so far, I 
only have gels with well focused bands for two times only. i have been 
getting a smear of stain (for enzyme activity) along the lane but no 
distict bands for my samples.  My samples are extracts of barley leaves. 
(barley leaves are homogenized in 0.01M phosphate buffer pH6.0, 
centrifuged at 500 x g for 20 mins, and supernatant are taken as samples 
for solulble peroxidase). Even though my gels did not show distinct 
bands for my samples, distinct bands are found for protein standards.
I am new with this and have no clue as to what to do next. If anyone 
could help me with this, I would really appreciate it.
Aye m.Tin

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