MacFS User wrote:
> > i'm trying to purify a HIS TAGGED protein on nickel-NTA RESIN, from
> qiagen. i'm having very little binding and very poor recovery . i need to purify under native conditions , does anyone have any suggestions please.
>
A colleague here does what you have asked about, so I am passing along
his suggestions:
1) check the pellet to see if you are really solubilizing the protein.
Try conditions like 50mm, 250mm, 1M NaCl, with/without detergent,
with/without betamercaptoethanol (do NOT use DTT - it binds strongly to
the NI column)
2)do not use DTT or EDTA if can avoid it. If you must use it to
resolubilize, then wash by diluting out, and spin several times before
loading onto the column.
3)try phosphate buffer or lower strength TRIS or HEPES for loading. In
some cases amines, including the buffers, can interact with the column
and prevent binding.
4)If you can't get the protein off the column, supplement with
imadazole, and try additional NaCl or detergent or glycerol to get the
bound protein to elute.
5)Try batch elution: empty the colum into a tube that can be gently
rocked, filter off the resin in the morning, measure protein in the
solution.
6)Presence of DNA or macromolecules may inhibit proper binding. Some
people treat with a small amount of DNase to chop up DNA into small
pieces. High viscosity is a problem. Large particulates, aggregates of
proteins should be spun out before loading.
7)If your protein is simply not soluble, then try putting your tag on
the opposite end, i.e. switch N term for C-term tag. Sometimes the end
which the tag is on makes a big difference in solubility.
8)As a last resort, if the protein simply will not elute from the resin,
strip the column with EDTA. This strips the NI plus the protein, but
you should be able to measure the eluate for protein content---it just
has a lot of NI in it. And the column can be regenerated.
9)The protein structure can be 'loosened - up' in 1 to 2 M urea or
guanidine, which really does not denature. Maybe even load the column
in this. But when proteins have been totally denatured then
everything tends to bind to the NI column.
Good luck. This info is second hand, but I have tried to accurately
pass my colleagues thoughts along. Tom McCloud SAIC/Frederick
Cancer Research
