Dear All,
I am working on protein 28 KDa, Construct has been made in pET28a vector with N terminal 6XHist tag, Protein expression has been optimised.
Protein is purifying nicely on NiNTA resin, as found on SDS-PAGE at expected mol weight 28-30 kDa.
Since the protein we want for crystallization and structure determination, We are applying it on Superdex 200 (HiLoad 16/60, 120 ml column) size exclusion chromatography, all protein is coming only around 45-50 ml, but on SDs-PAGE single protein around 30 kDa.
So it seems oligomerization.
we tried Size exclusion S200 with or without concentrating the NiNTA fractions 1mg/ml or around 10 mg/ml, with or without cleaving His tag, in all cases, protein is showing peak at 45-50 ml.
our Size exclusion buffer is 50 mM Tris pH 7.5, 500 mM NaCl, 10 % Glycerol, 1 mM DTT.
How to proceed? what should we try for getting the monomeric protein.? any suggestions? can we still try to set crystallization?
