From: John Philo <jphilo@earthlink.net>
  To  : 'Arturo J. Morales' <art@scripps.edu>
  Date: Wed, 20 May 1998 20:34:57 -0700

RE: lowest OD/conc...

Art,

First, you certainly don't need an absorbance as high as 0.5 to get good
results.  You should be able to work at 0.1 AU or less if you include
quite
a few scans in the analysis using either DCDT or my SVEDBERG program.

Furthermore, if you do absorbance scans at 229 nm instead of 280 you
will:
1) increase the absorbance by 10-fold or more for something without any
TRPs
2) get excellent signal/noise because the lamp puts out a lot of light at
this xenon emission line (assuming your lamp is clean!).

Using 229 nm I have gotten useful data on as little as 4 micrograms of
protein.

As regards interference data versus absorbance, in my experience I get
better signal/noise for low concentrations using absorbance at 229,
especially for small proteins where the slow sedimentation means that the
slow scan speed in absorbance is not much of an issue.

Further, given the small size of your peptide you will almost surely
want to
run at 60K rpm, and due to timing problems the noise level of the
interference data goes up about an order of magnitude at rotor speeds
above
50-55K.

Lastly, I disagree with Walt Stafford---your peptide is not too small for
good results from velocity, provided that you run it at high speed.

Good luck,

John Philo
Alliance Protein Laboratories


-----Original Message-----
From: Arturo J. Morales [art@scripps.edu]">mailto:art@scripps.edu]
Sent: Wednesday, May 20, 1998 2:16 PM
To: RASMB@bbri.harvard.edu
Subject: lowest OD/conc...



Hi!

I have a 110aa peptide with no TRP's that I'm interested in calculating
its
molecular weight and interactions in solution... but since the instrinsic
abs is so low, I have a hard time purifying enough of it to get an OD of
>0.5 (I'm at .2-.4 right now, but that's using my whole sample from a one
liter prep :)  Is anyone experienced in the limits of detection for a
clean
sedimentation velocity run?  I'm not that familiar with the nuances of
interference optics, but we have an XL-I... so maybe that's an option...

thanks in advance!

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