From: Arturo J. Morales <art@scripps.edu>
  To  : rasmb@alpha.bbri.org
  Date: Mon, 04 Jan 1999 16:35:51 -0800

Interesting problem...

Hello all,

I have an interesting problem and I hope that anyone out there with more
expertise than me in AU can provide some insight... this list has been
great so far at helping me learn how to use the XL-I mostly on my own
(since I wasnt the one going to the Beckman training in our lab :)

Ok, here's the problem:

I have a protein-RNA interaction that I'm trying to quantify... I know it
binds... Kd in the low nm range...

If I do a velocity run on the RNA, I get about 3S which assuming vbar of
.53 gives me MW of ~25Kd (just about right for tRNA)... so far so good... 

If I do the same on the protein, I get about 2S, which is about 24Kd using
a vbar of .7595 (calculated from composition).  that works well too...
since it is expected to be a dimer (12kd monomers)

The problem arises when I mix them both... I end up getting about 2.5S-2.8S
(or so, I need to refine that part).  I know this is happening because now
the vbar for the complex is somewhere in between .53 and .75, of course,
depending what vbar I use, I can get any MW I want... which is not really
that useful... My main question is, how can I approach this problem?  I
would like to get stoichoimetry (which I believe is 2:1 monomer:rna, but it
could be 2:2...)  Is it acceptable to show the shift in S without
estimating MW? Can I determine vbar directly for a complex? what is the
best way?

I would appreciate any comments 

THANKS!

Art

----------------------------------------------------------------------
     Arturo J. Morales    (RPI
'94)             art@scripps.edu
   Department of Biology - Massachusetts Institute of Technology &
  Skaggs Institute for Chemical Biology - Scripps Research Institute
                   http://schimmel.scripps.edu/~art