[RASMB] smaller protein size than expected

[Cole, James]

Dear Fang
That is a big discrepancy.  What are the confidence intervals on the molecular mass?  Because you are spinning somewhat slowly for a protein of 16kDa, the confidence intervals could be pretty broad. At 25K the value of sigma for this protein is only about 1.2 cm^-2  and goes to about 2.4 cm^-2 at 35K. I would suggest that you spin this sample to give a range of sigma about twice what you have done.
It is also possible that the mass is off because this protein has an anomalous value of v-bar, but I'm not aware of any case where the error is this large. What v-bar are you using and how did you calculate it?

Given that you have checked for proteolysis after the measurement, the gel filtration experiment does not help to explain this error. You can get apparent low molecular weights on gel filtration if your protein interacts with the matrix so that elutes later than predicted based on stokes radius.

Hope this helps.
Jim Cole

-----Original Message-----
From:	rasmb-bounces at rasmb.bbri.org on behalf of fang.yi at yale.edu
Sent:	Sun 11/12/2006 2:04 PM
To:	rasmb at server1.bbri.org
Cc:	
Subject:	[RASMB] smaller protein size than expected

hello all,

I recently did sedimentation equilibrium runs on a protein (monomer size: 16
KD), at 3 concentrations (260, 130, 52 u M) at speeds of 25, 30, 35000 RPM.
Using Heteroanalysis, I could only fit the data into an ideal solution model,
with a monomer MW of 10KD.  Also, this protein runs at a MW lower than 
expected
on the gel filtration col also. SDS gel indicates it's the right size and not
degraded after the measurements.

Anybody has any suggestions why this protein appears to be such a "smaller"
protein?

Thanks a lot,

Fang Yi
Postdoc Fellow
Yale University
Molecular Biophysics and Biochemistry
New haven, CT 06511
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