Help! Protein oxidation on Nickel column!

Rajesh Krishnamurthy krishnam at umbc.edu
Thu Jun 15 20:35:00 EST 1995


Most of my experience has been with copper columns. I know it is not 
correct to extrapolate results from copper to nickel but I cannot but help 
make this observation.

When we passed BME through the copper column, it reduced the copper in 
the column and this reduced copper came off the column. This did two 
things - (i) made copper an impurity that was not desired and (ii) this 
reduced form of copper managed to generate hydroxyl radicals. We too 
observed loss of activity in our column, and presume that the hydroxyl 
radicals were responsible for that. 

BME played the role of a reductant in our case. I am not aware of 
anything that would prevent BME from reducing nickel, but have not 
conducted experiments with nickel. Forms of nickel having valence other 
than 2 are rare I admit.

The other transition metals such as Iron, copper and zinc do form their 
reduced states, and these out-of-ordinary states have been implicated in 
reactions leading to formation of hydroxyl radicals.

Basically what I am trying to say is that you cannot rule out damage 
mediated by metal, which we very often ignore, and which is possible in 
IMAC columns if conditions are right.

> unless you can't add BME to your column run for a reason related directly
> to your protein, you should add it to your column binding/washing and all
> elution buffers.  while DTT will destroy the column, BME has no ill
> effects on the column.  i use it at 5mM and it seems to remove any
> disulfide bonding problems.
> 
> good luck,
> 
> eric
> 
> -- 
> "i don't know what caffeine does for you, but i'm pretty sure that without it your head caves in."
> 
> eric c. anderson                                 anderson at pharmdec.wustl.edu
> dept. of molecular bio. and pharm.               (314)362-3963 (lab)
> washington univ. school of medicine              (314)362-7058 (FAX)
> 660 s. euclid box 8103                           
> st. louis, mo 63110
> 
> for a comprehensive list of bike related web pages... http://pharmdec.wustl.edu/~anderson/anderson.html
> 
> 

**************************************************************************
A reasonable man tries to adapt himself to the world. An unreasonable man
tries to adapt the world to himself. So it is quite obvious that all
progress depends on the unreasonable man. :-)

quotes from the graves - G.B.Shaw
************************************
Rajesh Krishnamurthy
E-mail: krishnam at umbc7.umbc.edu		Tel: (410)247-8175
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