We have a protein that is soluble when bound to the column but precipitates at the high protein concentrations and high pHs required for all of the coupling reactions. We were thinking of labeling it with Alexa Fluor 488 while on the column. Anyone done that?
I haven't tried it, but it might work. Another option is to label the protein at a lower concentration at which it is soluble, then remove the excess label by rebinding the protein to the column and washing the unincorporated label out.
Well I just tried it and it worked!! our troublesome protein is not soluble- at any reasonable concentration- in any solution above pH 3 EXCEPT when it is bound to a nickel-NTA column. I washed a one ml Histag column containing my bound protein extensively with high and low salt and then bicarb coupling buffer; and then applied a ml of reactive Alexa 488 dye (invitrogen) in coupling buffer and let it sit stoppered for an hour. After washing, I was able to elute fluorescent protein with 1M imidazole -and the second 1 ml fraction was clean by SDS-PAGE-and bright! (I remove imidazole by dialysis..in 100 mM acetic acid.).
I have to add that the labeling efficiency was low. When I repeat, I will rock the resin containing bound protein with the reactive dye for a few hours, rather than just running it into the column and letting it react with the protein there.
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