Dear All,
I am trying to generate my protein with Se-Met incorporation.
The purification protocol involves Ni-NTA followed by anion exchange followed
by gel-filtration. The native protein behaves well and on gel filtration there
is very little protein eluting at the void (s-200 column) with vast majority as
monomer.
However using the same protocol with the Se-Met protein 95% of the protein
elutes at the void volume but this soluble aggregate protein still behaves well
and can still be concentrated to 10 mg/ml without any visible precipitates. My
protein contains disulphides and thus I have not used reducing agents in my
purification buffers. Purification takes about a day and a half.
Could anyone give me advice on increasing the proportion of monomeric protein.
Thanks in advance,
Gordon
Buffers:
Ni-NTA: A: 50 mM Tris pH8, 500 mM NaCl
B: A + 300 mM Imidazole
Q column: A: 25 mM Tris pH 7.5 at 4oC
B: A + 1 M NaCl
Gel filtration: 50 mM Tris pH 8, 200 mM NaCl
M. Gordon Joyce,
Visiting Fellow,
Structural Immunology Section,
Laboratory of Immunogenetics,
NIH/NIAID,
12441 Parklawn Drive,
Rockville,
MD 20851
Phone: 301 594 0242 Office
301 496 3792 Lab
