Hello Crystallographers, I am trying to express and purify a soluble domain of a membrane protein for crystallization. The amino acid content is as below Ala (A) 12 13.8% Arg (R) 10 11.5% Asn (N) 2 2.3% Asp (D) 8 9.2% Cys (C) 1 1.1% Gln (Q) 1 1.1% Glu (E) 4 4.6% Gly (G) 16 18.4% His (H) 3 3.4% Ile (I) 0 0.0% Leu (L) 3 3.4% Lys (K) 1 1.1% Met (M) 1 1.1% Phe (F) 2 2.3% Pro (P) 6 6.9% Ser (S) 11 12.6% Thr (T) 1 1.1% Trp (W) 1 1.1% Tyr (Y) 1 1.1% Val (V) 3 3.4% I could purify the protein using IMAC to 95% purity, how ever, I had to elute with very high concentration of imidazole 2 M, still some protein is attached to the beads as I could observe on the SDS PAGE.
I concentrated the protein to 25 mg/ml of 3 mls and on performing SEC, I found the protein to be in the Void fraction of SEC 200. Any expert advice on how to optimize this purfication and why it is still attached to the beads? thanks in advance Anita
