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
