Hi Anita, Try to lower the ph of binding buffer if your protein allows also you may lower the concentration of salt like nacl if adding into the buffer also try to reduce the imidazole concentration of your eluted fraction gradually by performing serial dialysis before SEC. Alternatively, you can try cobalt IMAC instead of nickel which may have lower affinty to proteins his tag. Also it may possible that your trucated construct may have lesser stability. You can also try to add some additives like glycerol to improve its stability along with some reducing agent like DTT or TCEP. Hope this may help your protein.
Shivendra On Feb 25, 2015 9:13 AM, "Anita P" <crystals...@gmail.com> wrote: > 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 >
