Is it possible, as I have heard about some DNA-binding proteins, that it is
at least partially intrinsically disordered (ID), which might change the
Stokes radius? You could cross-check by SDS-PAGE or SEC (ID's run larger).
CD, with careful protein quantification, might also do the trick. ID itself
might also change the dn/dc value, perhaps due to hydration (about that I am
not sure, though--I have read that this has an effect on PSV for AUC,
though).
Jacob
*******************************************
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
Dallos Laboratory
F. Searle 1-240
2240 Campus Drive
Evanston IL 60208
lab: 847.491.2438
cel: 773.608.9185
email: [log in to unmask]
*******************************************
----- Original Message -----
From: "Engin Ozkan" <[log in to unmask]>
To: <[log in to unmask]>
Sent: Friday, March 27, 2009 9:57 AM
Subject: Re: [ccp4bb] off topic - static laser light scattering
> Dear Tassos,
>
> Your assumptions are right, if (1) your dn/dc is accurate, or (2) your
> machine is calibrated. We recently measured a protein of a similar size to
> yours, and when a 700 Da ligand was added to the buffer, the measured
> protein mass was increased accordingly. So MALS can be pretty accurate.
> For our dn/dc, for pure proteins, we always use 0.185 (not 0.19). For
> sugar groups, we assume a dn/dc of 0.14, and estimate a mass-averaged
> value for the glycoprotein (usually somewhere between 0.175 to 0.18). For
> DNA and RNA, the values will be different, again.
>
> You may also realize that by changing a simple calibration constant, you
> can modify your measured molar masses anyway you want. It may be time for
> a recalibration (it is not difficult, you can do it yourself). We tend to
> regularly run BSA, and see if everything is as expected with our
> equipment.
>
> Good luck,
>
> Engin
>
> Anastassis Perrakis wrote:
>> Dear all,
>>
>> The MALLS instruments on-line with an FPLC and with an RI detector,
>> should provide an 'absolute MW', shape independent,
>> and indeed in our hands they do well. Until yesterday, where a 21kD
>> protein pretends to be 25 kD. We did the mass spec
>> anyway, and its 21kD as we expected to the residue, but I am still
>> puzzled by that result.
>>
>> One central assumption for the MALLS formulas, is that dn/dc, the
>> specific refractive index increment, is constant for unmodified proteins,
>> made by aa with no sugars etc. Literature suggests dn/dc values for
>> proteins to be constant and between 0.189/0.190 is a good value,
>> with minimal buffer dependence for aqueous buffers with 'the usual'
>> salts.
>>
>> I am a rather bad physicist, but my reading tells me that dn/dc, and thus
>> light scattering, depends to the "laser-light induced dipole in the
>> molecule". Is there any reason to believe that in theory a molecule with
>> a very particular charge distribution (eg a small DNA binding protein
>> which is already a 'dipole') would have significantly different dn/dc
>> values? Is anyone aware of such an experiment? Literature searches were
>> in vain ...
>>
>> Best -
>>
>> Tassos
>
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