histag elution alternative
malexeye at jaguar1.usouthal.edu
Sat Nov 18 20:04:27 EST 2000
I just browsed through "methods-reagents" archive and noticed postings on Ni-NTA and DTT. Although I am not a chemist and so far I was reasonable enough to follow the manual and not discover the weel with DTT, I thought I'd put in my $.02 worth.
For a long time I had suspected that bown stuff is in fact NiS, Ni sulfide, not necessarily a reduced form of Ni. Dima's experiment had only confirmed my suspicion. Most sulfides with some exceptions (exceptions being Na, K, Ca, etc) are insoluble. Sulfides if Ag, Cu, Fe are black, so it is conceivable that Ni sulfide would appear brown, especially at LOW concentration of Ni as it is on column. Treatment with strong acid (as is in the case of HCl) should be able to convert NiS to NiCl2 according to NiS +2HCl -> H2S + NiCl2.
The only problem with this is that DTT is not H2S. However, chemically it is essentially two molecules of H2S linked by replacing one hydrogen in each with organic radical HS-R-SH. To me, it is conceivable that DTT has some residual acidic properties (H2S is a weak acid). Therefore, the product on the column is more likely to be
S-R-S where R is -CH2-CHOH-CH2-CH2-
which upon treatment with HCl should react essentially as NiS releasing DTT and forming NiCl2 with Ni conceivably retained on the column by NTA.
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