renaturation

Celeste Wagner celestem at dna.bchs.uh.edu
Thu May 22 09:49:06 EST 1997


hey guys,
I have been working on isolating a novel protein and have had somewhat
confusing results.  After several purification steps, I isolate a
relatively pure protein at 50K, from a coomassie stained gel.  When I
then run this sample through a molecular sieve column, enzyme activity
isolates at 25K.  I'm wondering if this could be a very strong
dimer,because it is a denaturing gel.
 I would like to run this on a thick polyacrylimide gel, cut out the 50K
band and attempt to renature it and assay for activity, (I'll probably
do this with the 25K region of the gel also although there is no visible
protein band there).  I believe that this will show if the 50K protein
has activity or if it is a dimer. If all else fails I will even add the
gel separated 50K to the 25K, and see if they are both neccessary.

My question is, does anyone know of a method or reference that could
give me and idea of how much protein should be used and how much is lost
as well as renaturation protocols?  I am in search of protocols!!


Thanks in advance,
Celeste Moore
Univ. of Houston



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