[RASMB] dn/dc

[Mattia Rocco]

It could also be that you don't have any major "error", and the value you're getting reflects the actual solution status of your protein.... Bear also in mind that SEC columns are not that great in separating, say, monomers from dimers if your sample is quite small, although the peak shape maybe should provide an hint if this is going on... How much are you away from the MS expected value?

Mattia


-----Original Message-----
From: Stephen McLaughlin [mailto:stephenm at mrc-lmb.cam.ac.uk]
Sent: Fri 3/18/2016 10:55 AM
To: Mattia Rocco; Andrew Leech
Cc: jphilo at mailway.com; 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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