western blotting - transferring a small MW protein

Ian A. York iayork at panix.com
Fri Mar 22 23:01:23 EST 2002


In article <john.hines-2203022205090001 at remote3-arap15.cis.yale.edu>,
 <john.hines at yale.edu> wrote:
>
>    What I'm looking for are recommended transfer conditions.  How many
>volts (or milliamps) for how long a time?  I don't want to blow my tiny
>protein through my nitrocellulose, but at the same time I want to get as
>much of it onto the membrane as possible.

It's been a while since I did anything much under 12 kDa, but I don't 
think I did anything particular about time or voltage.  I did use a 
different transfer buffer, but I can't remember how it was different.  
It's in the Harlowe/Cold Spring Harbor manual Antibodies: A Lab Manual.  

You might try PVDF as well as nitrocellulose; I think we had better luck 
with binding to that.  You might also try putting two layers of 
nitrocellolose behind the gel just to see if stuff is going through.  They 
make nitrocellolose with a 0.2 pore size, if it's a problem.

But in general, for a 12 kDa protein, we don't do anything special, and it 
works fine.

Ian 
-- 
    Ian York   (iayork at panix.com)  <http://www.panix.com/~iayork/>
    "-but as he was a York, I am rather inclined to suppose him a
     very respectable Man." -Jane Austen, The History of England




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