Pamela Beckmann wrote:
: In a DENATURING (6M) urea prep of the extracellular domain of a His tagged
: protein (lots of Cys) expressed in E Coli, much of the protein stubbornly
: refuses to elute off the nickel resin.
:: We have tried the obvious, like low pH (3), EDTA up to 100mM, 6M guanidine
: Triton up to 100%, and even Acetonitrile - in case the reaction was a
: hydrophobic one. In all cases there was always some bound protein left on
: the resin after treatment (as ascertained by running a small sample of the
: resin on an SDS gel and Coomassie staining).
:: Has anyone had a similar problem? Any ideas will be welcome!
Everyone has similar problem which is not a problem, really. Whenever
crude concentrated protein mix is run over _any_ solid phase, there is
some "non-specific" binding. Some of that non-specific binding is so
strong as to become "irreversible". No matter what chromatography
you do and what elution conditions are, running SDS page of
post-elution "leftovers" will always give you some protein (that's why
top part of heavily used columns turns brownish over time). Just
disregard it. The fact that _some_ of your protein elutes with 1% SDS
at ~ 100C but does not in 6M guanidine simply says that the binding
mode is hydrophobic and fairly strong. The fact that EDTA and pH3 do not
elute it says that the binding mode has nothing to do with metal
Ignore it and use that protein that is elutable by imidazole/EDTA -
the mode of binding on which your chromatography is based on.
Regenerate the column with 6 M GuHCl after use (_always_ a good idea!)
and discard after 2-5 runs (or, if you are paranoid, regenerate by hot
1% SDS - Sepharose withstand this just fine).
But... You mention lots of cysteins. Under denaturing conditions in the
absence of reducers they will form S-S bridges and cross-link your
protein into multimers. Because of the cooperative nature of "non-specific"
interaction with the column, those will be very hard to elute. On the other
hand, good reducing conditions are incompatible with "normal" IMAC.
You might, however, try eluting in the presence of 100 mM DTT (nickel
will be reduced too, so you will need to strip/recharge the column
afterward). I'd try this mix: 50 mM EDTA, 100 mM DTT, 6 M GuHCl, pH 8.0
at room temparature (or even 30-50C) - this takes care of all modes