histag elution alternative

Richard P. Grant rpg14 at yahoo.co.uk.invalid
Thu Nov 2 03:25:16 EST 2000


In article <8tq8il$8nq$1 at news.doit.wisc.edu>, 
klenchin at facstaff.REMOVE_TO_REPLY.wisc.edu (Dima Klenchin) wrote:

> The nickel is gone. We are talking about _recharging_ and that 
> necessarily implies stripping the original, fully or partially reduced,

>From Cornelius' posting, that's not that crystal clear.   I'm not sure 
whether he reduced it in the nickel-bound state or not.  It would be 
interesting to take some resin, treat it with EDTA (which seems to strip 
the nickel), then add DTT.  And, of course, compare with non-EDTA 
treated.  It looks like in the presence of DTT the nickel is reduced 
(well, duh) to a state that not just makes the Ni2+ fall off (because in 
the absence of DTT it can be recharged with NiCl2) but causes something 
else to happen - maybe Ni+ is strange, hmmm what about Ni-??  (thinking 
aloud here).  The other thing is, given that the nickel is resistant to 
10 mM bMe, and your point about the reducing potential of the cell, 
maybe the change wrought by DTT is chemical rather than oxidative?   
Sulphonation, perhaps?  Maybe someone who has time to waste (!) could 
check some redox potentials . . .

> stuff. So the only things left are agarose, NTA, and linker arm. I am 

NTA is 'what' triacetate?   I mean, what exactly is there?

Just looked it up.  Nitrilotriacetic acid.  Hmm.  What's that nitrogen 
doing?

> :> Anyone wants to bet? I say that reduced Ni-NTA agarose 
> :> can be stripped and recharged w/o a problem. 

Just noticed this again - you seemed to be talking about the resin in 
the presence of nickel, unless the above was just a typo?

> he got. I have not seen any similar manufacturer's claims. To the 

We got our wires crossed there; I apologize.

> contrary, they say that after several runs with a lot of E.coli 
> stuff, the column may turn brownish (can't be any different since 
> reduction potential of E.coli cytosol is very high), in which case it 
> _can_ be stripped and recharged. 

<aside - Qiagen touting the reusability of their system?  wow, it must 
be indestructable>

As above, this  points to a chemical reaction . . .  maybe?

> There, Richard, I suggest a book under $20 from amazone.com? :-))

Mwah hah hah.  As if post-docs over here aren't skint enough :-/

I've a better idea.  I'll suggest some experiments and you can report 
back with the findings.  :)  Alternatively, we kidnap a Qiagen scientist 
and, eh, *extract* the information we need.  :-))

r

-- 
Richard P. Grant                                     MRC Lab of Mol Biol
rpg 'at' mrc-lmb.cam.ac.uk   http://www2.mrc-lmb.cam.ac.uk/personal/rpg/
         'I think I speak for everyone here when I say, "Huh?"'






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