[RASMB] Velocity Experiments

John Philo jphilo at mailway.com
Mon Oct 21 13:14:00 PDT 2002


Rekha,

In part you have answered your own question: if you indeed get an excellent
fit as a single species, as you said, then the data is telling you the
overwhelming majority of your sample is the monomer, and it is expected that
you would have difficulties fitting as multiple species.

There are two fairly simple explanations for why oligomers might not be
seen:

(1) You didn't say how large an oligomer you think this might be, but since
you are seeing it by AFM I am guessing it is quite large. Thus it probably
sediments MUCH faster than the monomer, and if so it will no longer be
present in the cell at the times when your monomer has moved substantially
away from the meniscus (already pelleted). For samples containing a large
range of masses, with DCDT approaches it may be necessary to analyze data at
different times in the run to get mass estimates for the different species
(analyze data early in the run to get the big things, and late in the run to
get the small ones away from the meniscus).

(2) Even though you can see them by AFM, the oligomers may represent only a
tiny fraction (by weight) of the total protein, and thus only a tiny
fraction of the total absorbance.

Some other points:

(1) This is an interacting system, but if you do a multispecies fit with
this approach you are assuming it is a MIXTURE of non-interacting
components. If, and only if, the kinetics of dissociation of the oligomer
are VERY slow (hours to days), it is okay to treat the monomer and oligomer
as separate components, and you should get the correct diffusion
coefficients and masses from the widths of the peaks in a g(s*) curve.

(2) A mass of 4 kDa for your monomer is really too low for highly accurate
results with DCDT approaches, particularly at 50K rpm rather than 60K. (See
my message of 9/23/02 in the archives.)

John Philo
Alliance Protein Laboratories

-----Original Message-----
From: rasmb-admin at rasmb-email.bbri.org
[mailto:rasmb-admin at rasmb-email.bbri.org]On Behalf Of rekha srinivasan
Sent: Friday, October 18, 2002 1:22 PM
To: rasmb at rasmb-email.bbri.org
Subject: [RASMB] Velocity Experiments


----------------------------------------------------------------------------
------
The older archived RASMB emails can be found at:
http://rasmb-email.bbri.org/rasmb_archives
and current archives at
http://rasmb-email.bbri.org/pipermail/rasmb/
Search All the Archives at:
http://rasmb-email.bbri.org/rasmb_search.html
----------------------------------------------------------------------------
------

Hi,
I recently did some velocity experiments (for the first time) with a small
peptide of 3935.5 Da mass.  This peptide forms spherical oligomers (examined
by AFM) within an hour of sample prep. The oligomers are stable upto 6 days
after which they assemble into chain like fibrils.
I wanted to get an approximate size of the oligomers and their percentages.
When I try to fit this data with DCDT+ (since it is supposed to be good for
small peptides, multi species samples) I get an excellent fit for single
species with M = 3700.  But everytime I try a multispecies fit it just gives
me wierd numbers....
What am I doing wrong?  Where are my oligomers?
Some trivial exprerimental details:
concentrations: 0.2 mg/ml and 0.5 mg/ml
buffer: 10 mM CH3COOK
rpm: 50,000
Thanks
Rekha

P.S: I would like to subscribe for the mailing list too!


Rekha Srinivasan
Graduate Student,
Department of Chemistry,
Case Western Reserve University,
2074, Adelbert Road, MSC 3N
Cleveland, OH 44106
Phone # 216-368-4476
Fax # 216-368-3006
email: rekhasun at hotmail.com

_________________________________________________________________
Surf the Web without missing calls! Get MSN Broadband.
http://resourcecenter.msn.com/access/plans/freeactivation.asp

_______________________________________________
RASMB mailing list
RASMB at rasmb-email.bbri.org
http://rasmb-email.bbri.org/mailman/listinfo/rasmb




More information about the RASMB mailing list