I would like to test the efficiency of desalting by a gel filtration column and I decided to check the desalting efficiency by monitoring removal of a dye through absorbance. Can anyone recommend a dye for such an application?
Hey Hello Pavel! Enjoying desalting proteins? I just saw that question and I couldn't resist at all. I have tried few experiments some long long times ago. The 2 main issues I found: (1) the low molecular weight molecule can interact with the high molecular proteins and (2) Few salts can also form some larger agglomeration on column. Using Fe(III) based salt could be messy and spread over a large range of elution volume...at least in my hand. I would try a copper based salt (blue-ish). Other salts or small molecules are possibles of course see:
https://en.wikipedia.org/wiki/Color_of_chemicals
For the high molecular component, you can look at Sigma; some fluorescent BSA for example is cheap...but BSA is quite an interesting protein as it binds to a lots of molecule as well... But do a quick search in Sigma catalog, and you could find other proteins that are covalently attached to a fluorophore or another probe. Another protein to possibly use from Sigma is cytochrome c (red naturally) but it maybe a too small protein for your method optimisation. Obviously I would avoid using overlapping chromophore....
Good luck Pavel,
T
great point! I guess this is why everyone checks desalting efficiency by BSA + NaCl. Indeed BSA will probably bind most of the dyes, since all dyes are hydrophobic and BSA will bind hydrophobic molecules non-specifically! I imagine some other non-organic, but absorbing salt will be best
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