[RASMB] Peptide sedimentation

Tue Jul 9 07:14:32 PDT 2013

Make sure that your initial absorbance in the AUC before you spin fast is
the same compared to the absorbance determined with a UV spec in a cuvette
(path length is different in the AUC). If the absorbance is different
between the two measurements, then you sediment big particles already during
the initial acceleration and you miss them in your scans.
Titus

-----Ursprüngliche Nachricht-----
Von: rasmb-bounces at list.rasmb.org [mailto:rasmb-bounces at list.rasmb.org] Im
Auftrag von Fasolini, Marina [Nervianoms]
Gesendet: Dienstag, 9. Juli 2013 15:42
An: Borries Demeler; Lake Paul
Cc: rasmb at rasmb.org
Betreff: Re: [RASMB] Peptide sedimentation

Thank you for your answers.
At 60000 rpm I only see the monomeric fraction of the peptide but I din't
see the aggregates. In literature it was mentioned a run at a very low speed
(4000 rpm) but in my hand, at 230 and 280nm, 50uM peptide, I had a problem
in the analysis of the data. Interference data gave some results, but not
very accurate. I think the speed is too low even if the aggregates are very
big. So I should run the experiments at an intermediate speed, adding salt
to the phosphate buffer and maybe acquire data also at 215nm.
Thank you

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

-----Original Message-----
From: Borries Demeler [mailto:demeler at biochem.uthscsa.edu] 
Sent: Tuesday, July 09, 2013 3:21 PM
To: Lake Paul
Cc: Fasolini, Marina [Nervianoms]; rasmb at rasmb.org
Subject: Re: [RASMB] Peptide sedimentation

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
> >
> >
> > _______________________________________________
> > RASMB mailing list
> > RASMB at list.rasmb.org
> > http://list.rasmb.org/listinfo.cgi/rasmb-rasmb.org
> >
> 
> 
> 
> -- 
> Lake Paul, PhD
> 
> Biophysical Analysis Lab
> Purdue Proteomics Facility
> 
> Bindley Bioscience Center
> Purdue University
> 1203 West State Street,
> West Lafayette, Indiana, 47095
> 765.494.4960
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