[RASMB] dn/dc

[Zou, Qin]

Not sure if the M.W. from SEC-MALS are larger or smaller than that from mass spec. it would be good to check if the retention time does not shift as a function of loading. If so, it may be due to some self-association on the column that could give a MW other than a monomer.

Qin

Pfizer Inc

 

-----Original Message-----
From: RASMB [mailto:rasmb-bounces at list.rasmb.org] On Behalf Of Stephen McLaughlin
Sent: Friday, March 18, 2016 4:56 AM
To: Mattia Rocco; Andrew Leech
Cc: rasmb at rasmb.org
Subject: Re: [RASMB] dn/dc

Dear Mattia, Andrew and John, 

The BSA standard recovery is as expected.  Hence, given that the Zn ions are not going to effect the dn/dc significantly, I think you are all right in suggesting that the error will be a compound of inaccuracy in dn/dc calculation coupled with lower light scattering for a small protein giving rise to an larger error that would be expected when analysing much larger proteins. 

Many thanks

Stephen   


On 17 Mar 2016, at 16:08, Mattia Rocco wrote:

> I agree with John regarding the dn/dc, but maybe the absolute calibration of the instrument could also be an issue... or the on-line concentration determination... as Andrew Leech pointed out this morning, do you recover the approximately correct molecular weight for, say, BSA?
> 
> Mattia
> 
> ________________________________
> 
> From: RASMB on behalf of John Philo
> Sent: Thu 3/17/2016 4:22 PM
> To: 'Stephen McLaughlin'; rasmb at rasmb.org
> Subject: Re: [RASMB] dn/dc
> 
> 
> 
> Stephen, I think it is unlikely the dn/dc for your protein differs by more than 5% from your calculation. If there is a "major" discrepancy then calibration is not the issue. 
> 
> John
> 
> 
> 
> -----Original Message-----
> From: RASMB [mailto:rasmb-bounces at list.rasmb.org] On Behalf Of Stephen McLaughlin
> Sent: Thursday, March 17, 2016 5:08 AM
> To: Mattia Rocco <mattia.rocco at hsanmartino.it>
> Cc: rasmb at rasmb.org
> Subject: Re: [RASMB] dn/dc
> 
> 
> 
> Dear Mattia, 
> 
> 
> 
> at the minute, it would be good to know if the dn/dc increases or decreases  and roughy how much. There is a significant discrepancy between the molar mass measured by SEC-MALS and the expected mass from mass-spec. 
> 
> 
> 
> Best 
> 
> 
> 
> Stephen
> 
> 
> 
> 
> 
> 
> 
> On 17 Mar 2016, at 09:53, Mattia Rocco wrote:
> 
> 
> 
>> Dear Stephen,
> 
>> 
> 
>> it likely will. But calculating a dn/dc is not that reliable anyway... and what's the level of precision you need, after all?
> 
>> 
> 
>> Best - Mattia
> 
>> 
> 
>> ________________________________
> 
>> 
> 
>> From: RASMB on behalf of Stephen McLaughlin
> 
>> Sent: Thu 3/17/2016 10:40 AM
> 
>> To: rasmb at rasmb.org <mailto:rasmb at rasmb.org> 
> 
>> Subject: [RASMB] dn/dc
> 
>> 
> 
>> 
> 
>> Dear all,  
> 
>> 
> 
>> I have a small three fingered Zn binding protein.  Will the presence of Zn ions affect the refractive increment that can be caluculated from sequence?
> 
>> 
> 
>> 
> 
>> Best 
> 
>> 
> 
>> Stephen
> 
>> 
> 
>> 
> 
>> ______________________________
> 
>> 
> 
>> Stephen McLaughlin, Ph.D.
> 
>> 
> 
>> Biophysics
> 
>> MRC Laboratory of Molecular Biology
> 
>> Francis Crick Avenue
> 
>> Cambridge Biomedical Campus
> 
>> Cambridge, CB2 0QH
> 
>> 
> 
>> Tel.: +44 (0) 1223 267891 (office)
> 
>> 
> 
>> Email:stephenm at mrc-lmb.cam.ac.uk
> 
>> _____________________________
> 
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