[RASMB] Peptide sedimentation

[Borries Demeler]

Dear Marina,
Lake's recommendations are right on target, I want to add one more:
You may want to test multiple concentrations to see if there is a mass
action effect. If your sample is self-associating in the concentration
regime of your test, you should see a shift of the s-value distribution
to higher s-values as you increase the concentration. Keep in mind that
your question about aggregation (or perhaps reversible self-association)
should be evaluated at the same concentration at which you want to use
the peptides. For example, if you plan to make a drug formulation at 5
mg/ml and you are wondering if the peptides reversibly self-associate,
it doesn't help if you measure it at 0.1 mg/ml at some low wavelength,
then you really should measure it at 5 mg/ml, since you cannot necessarily
extrapolate to the higher concentration from your measurement at low
concentration. While this approach may work if your peptide is indeed
irreversibly aggregated, the picture would probably be very different if
your peptide reversibly self-associates. So always check the sample at
the concentration of actual interest to get the actual size distribution.

Regards, -Borries


On Tue, Jul 09, 2013 at 08:30:58AM -0400, Lake Paul wrote:
> Marina,
> First I would make sure you have at least 50 to 150 mM NaCl in your
> buffer. I would also spin at 60,000 rpm if possible. If you can't then
> do it at 50, 000 rpm for a longer time. The wavelength is dependent on
> whether you have W, F or Y in your peptides. If you do then you can
> use 280 nm. If you don't then 220-230 nm will be your best option. The
> only catch is that you have to make sure your buffer does not absorb
> in that wavelength range. Fortunately, the masters of the AUC did
> these experiments for us and there is a list of acceptable buffers you
> can use. Phosphate is a good one at that wavelength. I would also
> search the archives of RASMB. I would treat it like a peptide like a
> protein in terms of concentration. 0.1 to 1.3 OD range or the range in
> which you see aggregation.
> This should get you started.
> Lake
> 
> On Tue, Jul 9, 2013 at 5:51 AM, Fasolini, Marina [Nervianoms]
> <Marina.Fasolini at nervianoms.com> wrote:
> > Dear All
> > I need to follow the aggregation of some peptides of about 4 KDa . I would
> > like to estimate the dimension of these aggregates.
> > Since my experience is mostly in protein sedimentation, I was wondering if
> > you have some tips for me. Which wavelength do you use for absorbance optic?
> > Which is the best concentration to run?
> > At which speed do you suggest me to run the experiment?
> > Any help will be appreciated.
> > Thanks in advance
> > Marina
> >
> >
> >
> > MARINA FASOLINI
> > Structural Chemistry
> >
> > Nerviano Medical Sciences http://www.nervianoms.com
> > Viale Pasteur 10
> > 20014 Nerviano - Milano
> > marina.fasolini at nervianoms.com
> > Tel. +390331581462
> > Fax. +390331581360
> >
> >
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> 
> 
> 
> -- 
> Lake Paul, PhD
> 
> Biophysical Analysis Lab
> Purdue Proteomics Facility
> 
> Bindley Bioscience Center
> Purdue University
> 1203 West State Street,
> West Lafayette, Indiana, 47095
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