Choice of resin for size exclusion chromatography - (Mar/14/2006 )
I've been having difficulty in purifying my protein which has a C-type lectin-like domain.
Mainly this protein was cloned to possess a His6x tag for the purification purpose from Ni column.
No matter what we've tried on the imidazole concentrations in equalibration/wash/elution buffers, the eluted materials never looked pure (still contained quite a few fairly visible bands by SDS-PAGE, presumably contaminants from the E. coli lysates) enough for subsequent experiments.
We had tried using gel filtration with Pharmacia's Sephadex/Superdex resin, however, our proteins now easily got stuck onto the column and almost impossible to get them off the column.
I had recently come across BioRad's polyacrylamide-based resin info and am wondering if any expert might have prior experience with it Bio-Gel P series resin for protein purification before. My protein's m.w. is around 110 kD, so would assume its Bio-Gel P-100 might be the choice to try. Any suggestion would be highly appreciated!!!! Many thanks!
It is strange enough that the protein would bind to a dextran/agarose based resin. Probably a long shot but using an appropriate ammount of NaCl (50mM to 0.5M) in the elution buffer has been shown to inhibit protein matrix interactions. Depending on your contaminant size, you could also choose a resin with a fractionation range smaller than that of your protein. This way your protein at 110 kDa is eluted in the void volume first and other contamiants get caught up in the matrix. Reverse size exclusion!!