I am using DLS measurements in order to calculate the second virial coefficient of a full-length mAbs in the presence of different buffer compositions and to do that, refractive-index increment for the protein/buffer pair (dn/dc) is required.
Have you some experience regarding that?
Particularly, I would to know if I can use the dn/dc = 0.185 g/mL that is usually suggested for globular protein also in my case, where the shape is not exactly globular. If I cannot, do I need to determine it by refractometry or there is a dn/dc that can be used for full-length antibody?
Thanks
I think you will find this paper useful:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3149238/pdf/main.pdf
My takeaway from it is that for larger proteins, the value of dn/dc falls within quite a narrow range, and depends on the amino acid composition rather than the protein shape.
The paper is really interesting: the dn/dc distribution for human protein is a gaussian (average=0.1888 ml/g devstd=0.003) that is more narrow for >100kDa protein, as in my case. However, they are calculated dn/dc, although measurements on two peptides suggested that experiment and calculation give basically the same value.
I will use SEDFIT calculated value for dn/dc, although the discrepancy between calculated and experimental value worry me... the dependence of the second virial coefficient by the dn/dc is strong.
As an additional comment, the paper (Zhao) referred to by Shapiro above, is excellent, IMHO. I also referenced the paper in in this earlier webinar which may be of use (registration required):
June 26th, 2012 Uses of an Abbe refractometer https://www.malvernpanalytical.com/en/learn/events-and-training/webinars/W120626AbbeRefractometer.html
You'll see references back to 1903 for this dn/dC value for proteins (slide 25 onwards)
For the amino acid chain, the dn/dc value can be calculated with SEDFIT; as the complete molecule contains may however carbohydrate side chains as well, please refer to the calculation by Pletcher C et al, Deglycosylated Prothrombin Fragment 1, J Biol Chem 255 (1980), 7433 - 8, where a dn/dc value of 0.000140 mL/mg is given for glycans.
Therefore, the total dn/dc = wt. % protein × dn/dc SEDFIT + wt % glycan × 0.000140
Dear colleagues,
When working with glycosylated proteins or glycoconjugates, I advice to use the mass spectrometry to determine to % of glycan part. The mass spectrometry is much more precise than SDS PAGE or AUC. With AUC the partial specific volume for glycosylated proteins is still an approximation.
The contribution of glycans is around 0.140 and not 0.000140. Still an approximation.
The best method is to use a refractometer.
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