"Dr. Artem Evdokimov" <eudokima at mail.ncifcrf.gov> wrote:
:> 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).
My sample size is something like 7-10 :-)
:> 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.
I disagree. If you use "just enough" column capacity, not
only it is not wasteful, but it frequently gives better recoveries
for problematic proteins. I was purifying a protein for the same
purpose (xtal), I had about 150 mg in 200 ml insect extract
loaded on two wide 7 ml columns. I got 120 mg after dialysis
and because I used steep 150 mM imidazole elution after rigorous
wash, no0t only the purity was good enough for crystals, but
a concentration was 8.5 mg/ml, good enough to go without
further concentrating step. The problem with this approach
is that it is painfully long and requires more careful optimization.
: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).
Sure, that's way to go if expression levels are not high enough
(low good/junk ratio).
:> :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)
I noticed that! One batch that was purified with AS pption had
different pH optimum than another batch that used Q column. Any
explanation for this effect? (Heterogenuity introduced
due to partial denaturation of complex proteins during pption
would be my first guess).
: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).
Yes, I believe 20V will do it even at low concentration of
chelator. I _was_ surprised to find that 3V of 20 mM do not
strip (100 mM did right away).
: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).
Don't know. I always use 2 mM in all extracts and never had
a problem in the cold room (I do regenerate/recharge columns
after each use though). To turn the column really brown, I had
to use 100 mM at rt for 30 min.
: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.
Thanks for the info about proline. I always put Gly, but perhaps
Pro is a better idea.
:Proteins are very different and one never knows what to expect.
:"Standard Protocols" barely ever work in protein purification on a large
:scale :)
Oh yes, sure. That's the beauty of working with them.
- Dima
