Jerry, In my experience with GE's HiTrap Chelating HP columns (which use the tridentate IDA linkage, rather than the tetradentate NTA), capture is usually quite good as long as the protein is well behaved and the tag isn't occluded. It is definitely possible to capture proteins at the level of 0.2-0.5 mg/L, as you are describing. With such low concentrations, you will want to perform the binding in column mode rather than in batch, however. You will also need to filter your medium through 0.22 um filters to avoid clogging the column with such a large volume.
The bigger problem may prove to be your medium. Most tissue culture media contain chelators that will strip your column; your particular medium may be OK, but you may want to contact the manufacturer (or just try it, with the understanding that it may not work). If your column is stripped, when you attempt to elute with imidazole (which normally turns your column a brighter blue), you will be able to see that the column is partially or completely white instead. If only a small portion of the column turns white (it will look like a white band at the top of the column), your capture may still work fine. If there is even a little bit of blue left, it may still be possible to switch to a larger column that retains enough nickel to capture your protein. If the column is totally stripped, however, you will probably have no choice but to concentrate and buffer exchange your medium. -Tom Cleveland Leahy Lab Johns Hopkins University School of Medicine On Mon, Apr 23, 2012 at 9:37 PM, Jerry McCully <for-crystallizai...@hotmail.com> wrote: > Dear All; > > This is a sort of naive question about the NI-NTA affinity > purification. > > > Is the Ni-NTA column from GE health able to capture proteins at > 0.2ug/ml to 0.5ug/ml? > > IF not, then it is necessary to concentrate the mammalian expression > supernatant. > > Thanks a lot and best regards, > > Jerry McCully > >
