[RASMB] smaller protein size than expected

[Schoenfeld, Hans-J.]

Dear Fank,
my recommendation would be to run gel filtration analysis with on-line static light scattering as kind of complementary experiment.
Best regards,
Hans-Joachim.
>============================================
>Dr. Hans-Joachim Schönfeld
>F. Hoffmann-La Roche Inc.
>PRBD-E, B93/5.44
>CH-4070 Basel
>Switzerland
>
>Tel. (+41) 61 688 28 95
>Fax. (+41) 61 688 90 60
mailto:hans-j.schoenfeld at roche.com


-----Original Message-----
From: rasmb-bounces at rasmb.bbri.org [mailto:rasmb-bounces at rasmb.bbri.org] On Behalf Of Fang Yi
Sent: Monday, November 13, 2006 12:36 AM
To: Cole, James
Cc: rasmb at server1.bbri.org
Subject: Re: [RASMB] smaller protein size than expected


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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