> In my experience, easily 50% of all 6-his tagged proteins
> elute at ~ 50 mM imidazole (pH varies 7.0-7.50, depending
> on protein).
Must be dependent on type of His tag (with preceding proline or without)
and on other characteristics of the protein. Our sample size is ~50
proteins during the last year, majority of which elute at 80-120 mM
imidazole (straight Hisx6 tag, no proline). If you add a proline before
C-terminal or after N-termina His tag, affinity normally goes up (most
likely due to the higher accessibility of the tag).
> I tried this a while ago and compared it to step elution.
> Purity-wise, step was much better _if_ it includes
> ridiculously thorough isocratic wash/elution step at
> ~ 10 mM imidazole (like, 200 ml wash of 2-5 ml
> column).
True. Unfortunately for the purposes of crystallization one needs to use
rather large columns (50 ml is our standard size) and it is somewhat
difficult to wash these with more than 8-10 c.v. Please note that if you
are using a *small* column with *large* amount of cell extract, you will
get higher purity because your target protein will be in excess.
Concomitantly, most of the target protein will be lost. Again, it all
depends on what purpose does the purification serve - we, for instance,
need to make lots of protein (40 mg is good, 100 mg is better) and thus
it is wasteful to use large sample/column ratios. In our case, IMAC is
always followed by several other purification steps so recovery is more
important than first-step purification ratio (though purity is, of
course, important as well).
> :Importantly, TRIS is NOT recommended for IMAC (in fact most amines would
> :interfere, including commonly used ammonium sulphate)
> Not for a single run. Once for me, it was really very convenient
> to do IMAC right after AS pption. So I did a trial run to see how
> His-tagged protein binds in 250 mM ammonium sulphate. It did
> bind perfectly well and eluted where it usually elutes. If I am
> not mistaken, Tris should be less reactive than AS. Sure, repeated
> use of a column with amines will shorten its life, but for a
> single run it's fine. Same for 5 mM DTT at +4C. Even chelators
> like EDTA and EGTA at ~ 2 mM do not interfere appreciably with
> binding (apparently, koff for Ni2+-NTA is small enough).
Very dependent on the circumstance. 300 mM ammonium sulfate inhibited
binding of at least 4 proteins that I have tried (I could have tried
more but this is a pointless excercise because we do not use
precipitation very often (bad for some xtals) and we don't have AS in
our buffers). I have once mistakenly used inhibitor tablets containing 1
mM EDTA (final conc.) and it has stripped my Ni-NTA column completely,
during the course of the load (500 ml over 25 ml column). IMHO 5 mM DTT
has turned the flow-through and some of the eluate brown due to
formation of nickel complex(es) (though some of the protein still was
bound to the column and eluted normally). TRIS normally is not a serious
problem, however in a delicate case like that reported in the original
note, it may be wise to minimize potential causes for trouble and that
includes all the points discussed above. It may be useful to insert a
proline residue, in order to maximize binding, or to grow the tag by a
few HIS residues, or to use an affinity sandwich, etc.
Proteins are very different and one never knows what to expect.
"Standard Protocols" barely ever work in protein purification on a large
scale :)
A.
--
|Dr. Artem Evdokimov Protein Engineering |
| NCI-Frederick Tel. (301)846-5401 |
| FAX (301)846-7148 |
|eudokima at mail.ncifcrf.gov |
|http://www.ncifcrf.gov/plague |
