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Help! My HisTag Protein won't come off Ni Resin

Tyson tyson at canada.com
Thu Jan 27 19:56:39 EST 2000


your problem is probably the cys residues which are keeping the protein
aggregated on the column, however you will always have a significant amount
of your protein left on the column....try the following protocol (maybe u
have already?)

-incubate the prep in 6M GnCl, NaP, Tris/HCl @ pH 8  for ~ 1 hour @RT

-cf prep & load on to the column (pre-equilibrated w/ GnCl !)

-wash w/ 10 vol. of this GnCl sol'n

-wash w/ 8M urea, NaP, Tris/HCl (pH8.0) until A280 of eluate <0.01

-wash w/ above sol'n @ pH 6.3 until A280 <0.01

-elute the protein w/ 2% SDS, 10mM EDTA, 0.1M NaP @pH7.0

.....using triton up to "100%" and concentrations of EDTA or reducing agents
>100mM will cause even more problems and leave even more protein on the
column.

....also you might want to try using DTT (<50mM)

hope this helps,

Tyson

"Pamela Beckmann" <pbeckma at nimr.mrc.ac.uk> wrote in message
news:v01530500b4b39f262dc2@[193.63.83.36]...
| 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
| HCl,
| 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!
|                                                     Pamela Beckmann, NIMR
|
|
| ---






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