From: Borries Demeler <demeler@bioc09.v19.uthscsa.edu>
  To  : Stephanie Morris <smorris@medusa.bioc.aecom.yu.edu>
  Date: Thu, 26 Jul 2001 13:30:43 -0500 (CDT)

Re: small peptides

Stephanie,

I think your best bet is to run as fast as possible (60krpm, aluminum
CP's) and to run as long as possible with a fully filled column.
If your goal is to measure the degree of heterogeneity, you will have
a hard time identifying that, since you will not be able to separate
very well, because of low s-values. If you have 1 or 2 distinct components
in the mixture, you may have some luck with UltraScan's finite element 
analysis or the finite element analysis program from Peter Schuck, 
if the residuals look fine for a one component model, you may be able
to deduce MW from your system. One of the difficulties you will face is
the lack of a stable plateau due to fast diffusion for the small peptides,
so dcdt and van Holde - Weischet will have difficulty with the data.

Equilibrium expt. are also an option, but you will have a hard time 
getting enough curvature in your equilibrium gradient. Try equil. speeds
between 40 and 60 krpm, and make a global fit of multiple loading 
concentrations and speeds. To make up for the shallow gradients, you
can load a slightly longer column, maybe 120-150 microliter. This will
produce more datapoints. They will come to equilibrium quite fast, since
the diffusion will be large for those samples.

While you can measure samples that small in the AUC, this size range is
not really the ideal range for the best data you can get with an AUC.

Good luck, -Borries
> 
> Dear RASMBers,
> 
> I was wondering if anyone has performed sedimentation velocity or
> sedimentation equilibrium experiments on small peptides?  Recently, I
> performed a sed velocity experiment with a peptide that is about 2,000Da
> at a speed of 55,000rpm.  The boundaries were very broad and difficult to
> analyze using programs such as DCDT+ and SVEDBERG. Does anyone have any
> suggestions for running sed. velocity experiments using very small
> peptides and for analyzing this data?  I'm also interested in performing
> sed. equilibrium experiments with this peptide. Are there any quirks or
> helpful tips for running these kinds of experiments?  Any suggestions
> would be greatly appreciated.
> 
> Thank you very much,
> 
> Stephanie
> 
> Stephanie Morris
> Albert Einstein College of Medicine
> 1300 Morris Park Ave.
> Bronx, NY 10461
> smorris@aecom.yu.edu
>