GFPuv Western Blot

The Grouchybeast thegrouchybeast at hotmail.com
Tue Jan 30 07:21:21 EST 2001


> Rainbow wrote:

> Recently I have expressed my GFPuv protein from clontech in E. coli
> TOP10F'. I could detected a band corresponding to 27kDa when I did
> Western blot. However, I also found an additional band which is around
> 50kDa, even though when I repeat the experiment again.

It would help if you could give a little more detail of the methods and
reagents.  E.g.

What are the conditions for the expression in the e.coli?
What antibody?
Reducing or non-reducing gel?
Was the sample purified in some way or whole lysate?
Did you run a control lane of non-transformed e.coli lysate?
Did you have a recombinant GFPuv control lane?
Are you getting good fluorescence?

The Living Colours Manual section II-G-8 mentions that GFPuv has a
greater tendancy to dimerize than GFP, so it's possible that you're
seeing the dimers on the Western.

> Kenneth

love
Anna
-- 
I see you standing there, far out along the way,
   I want to touch you but, the night becomes the day.
I count the words that I am never going to say,
   And I see you, in midnight blue.    (ELO - Midnight Blue)






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