[RASMB] smaller protein size than expected

[Fang Yi]

Dear Jim,

Thanks for your prompt reply. For the analysis, I used the default vbar 
and density values of 0.73 and 1 respectively.  At 35K, the value of 
sigma for this protein is only 1.44 cm-2 (ideal  model). The highest 
speed recommended to run on this specific centrifuge is set to 40K.  I 
completely agree with you that gel filtration results doesn't help much 
in this case considering the possibility that the protein might interact 
with the matrix. Actually that's  why we decided to do AUC. 

Fang

Cole, James wrote:

>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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