[RASMB] peptide concentration

[Borries Demeler]

Hi Christine,

In general, high concentrations can give rise to steep gradients at
the bottom of the cell, which may introduce Wiener skewing (an optical
distortion of the gradient) once the slope gets too steep. But for
molecules as small as your peptides you shouldn't have too much of a
problem with gradients that are too steep, even at the highest speeds
possible with the XLI (which you will have to use to characterize
molecules that are that small, and if you run long column expts. you
could exclude some of the steeper regions at the bottom). 

For best results I would probably run a long-column velocity experiment
and fit the approach-to-equilibrium data using finite element models
to get molecular weights from s and D. Short column experiments should
give you quickly equilibrating gradients you should be able to fit for
MW directly.  Another question you should consider is if there is some
concentration dependent non-ideality that may have to be considered at
such high concentrations. This may require fitting the virial coefficient.

I am not sure how stable your protein is, but in general you should be 
able to recover your sample without any problem. The only time this may
be difficult is if the sample actually pellets out and sticks to the 
wall of the centerpiece, making recovery less efficient.

-Borries

> 
> Hello
> 
> I basically have 2 questions.  How high of a concentration of peptides can
> you run with interference optics in a sedimentation velocity experiment? 
> And what are the odds of recovering usable peptide after the run?
> 
> For a little background, I ran some peptides (12-33 aa in length, no 
> tryptophan or tyrosine) using interference optics in an SV run (400ul
> sample) to check for self-association of the peptides.  I ran the samples
> at a concentration of 4 mg/ml to mimic what they have used in experiments
> before as well as conserve their peptides.  However, there was no evidence
> of self-association.  Turns out they have seen aggregation using AFM at a
> concentration of 40 mg/ml.  Now they would like to confirm this by AUC. 
> And they would like to get their sample back.  Hence my 2 questions.
> 
> Any advice would be appreciated.
> Christine
> 
> 
> _______________________________________________
> RASMB mailing list
> RASMB at rasmb.bbri.org
> http://rasmb.bbri.org/mailman/listinfo/rasmb
>