Hi all,
I am thinking about other approaches than SEC to separate protein-dye conjugates from free fluorescent dyes, like Alexa, Cy and Atto dyes. Does any of you have experience with that? Will the dyes stick to the matrix like it usually happens in SEC using Sephadex G-25? Or will they wash off easily?
Thank you in advance.
Best regards,
Gustavo
Hi Gustavo Avelar Molina . The problem with using ion exchange chromatography to separate free dye from labeled proteins is that dyes and proteins have different charges. So the same protocol can't be used to separate all proteins from free dyes like in SEC.
Dialysis would be an alternative.
I think gel filtration is the best method. Dialysis doesn't seem to work that well for some dyes, although it can be useful to reduce the concentration of free dye before gel filtration. Cleaning the gel filtration column to get the residual dye off can be avoided by using disposable desalting columns, such as PD-10.
Riaz A. Khan How would you wash using DMSO? Manually?
Steingrimur Stefansson I am only having trouble with one protein, so it is not important that the protocol cannot be used for other proteins.
Adam B Shapiro I think the biggest problem for me is that my protein interacts very strongly with the Sephadex G-25 resin in a HiPrep 26/10 column, so I normally recover only about 5% of the total protein, and this fraction is in reality badly-folded protein that is not functional (exactly because of that it can be separated). It is unclear when the "good" protein is eluted. Even if I knew, there would probably be free dye contaminating it. But I get your point of sticking with SEC, so I might try increasing the salt concentration of my elution buffer (currently I'm using 25 mM MES pH 6.0 + 100 mM NaCl).
Best regards,
Gustavo
Yes, you can wash manually, and flash run, VLC (Vacuum Liquid Chromatography) method, and also with nitrogen-pressured DMSO flowing off the column.
Gustavo, if Sepharose based gel filtration resins (e.g. Sephadex as in PD-10 columns) won't work for your problem, acrylic resins (e.g. Sephacryl) might be an option worth trying.
If you really want to develop an ion exchange procedure, you could try a standard anion exchange resin (e.g. Dowex), as you want to remove a small molecule. You don't need the pores of a resin that is intended to bind proteins.
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