Hi, Tassos,

It appears I am no better a biophysicist than you are on this one (I can 
see my Scattering instructor from grad school disapproving of me right 
now, he reads this bb). I cannot connect how the laser-induced dipole 
and concentration-dependence of refractive index of your sample would 
depend on each other (any biophysicists out there?).

But I still thought I should mention a few practical points that might 
help: dn/dc depends a bit on wavelength, so not all literature values 
might be the right ones for you (we use 0.185, because someone at Wyatt 
must have told us so).  Second, if you really want to know your dn/dc, 
measuring it might be the thing to do. You probably already have an RI 
detector (Optilab from Wyatt?). If you can get yourself a medical-grade 
syringe injector, you can inject your sample into the RI detector 
(otherwise it is very hard to do so) at multiple concentrations, and 
calculate for yourself a dn/dc. Obviously, you have to (1) have decent 
quantities of material to waste, and (2) be very accurate with your 
dilutions/concentrations. Wyatt also sells something that might help you 
do this experiment, that might be the easier way.

Third, sometimes, inspection of the Debye plot and removing outlier 
several angles from the plot helps improve accuracy (and precision, but 
that is not always a good thing). Especially smaller angle measurements, 
which seem to sometimes cause trouble in our hands (We usually use 
detectors 10-17 on the 18-angle Wyatt MALS machine). This sounds like 
throwing away data, but it is more like throwing away outliers and 
inaccurate data (Wyatt claims only three data points is enough to get a 
good molar mass, in most practical cases). Use your statistical judgment:)

Good luck,

Engin

On 3/30/09 1:16 AM, Anastassis Perrakis wrote:
> Hi Engin -
>
> On Mar 27, 2009, at 15:57, Engin Ozkan wrote:
>
>> Dear Tassos,
>>
>> Your assumptions are right, if (1) your dn/dc is accurate, or (2) your
>> machine is calibrated.
>
> indeed!
>
>> 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).
>
> yes, thats what most people use. Some literature claims 0.182 ... 0.19 
> is an old value, indeed.
>
>> 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.
> yes.
>>
>>
>> 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.
>
> yes, we did re-calibrate. I was very puzzled since we had very 
> accurate results before as well.
> my question was more towards trying to understand my protein a bit:
> the fact that it appears to have a strange dn/dc value, would it 
> suggest that it is a
> very strong molecular dipole?
>
> I better solve the structure ;-)
>
> Cheers - Tassos
>
>>
>>
>> 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
>>
>
> *P** **please don't print this e-mail unless you really need to*
> Anastassis (Tassos) Perrakis, Principal Investigator / Staff Member
> Department of Biochemistry (B8)
> Netherlands Cancer Institute,
> Dept. B8, 1066 CX Amsterdam, The Netherlands
> Tel: +31 20 512 1951 Fax: +31 20 512 1954 Mobile / SMS: +31 6 28 597791
>
>
>
>