Protocol for GSH affinity matrix

WS via methods%40net.bio.net (by novalidaddress from nurfuerspam.de)
Sat Mar 26 12:46:04 EST 2011


Dear Jose,

You probably came from this post from the archives:
http://iubio.bio.indiana.edu:7131/bionet/hypermail/methds-reagnts/1996-May/044702.html

1ml of resin should bind 10mg of protein or more; how many grams of
protein do you want to purify?

I doubt you will get a homemade resin cheaper than GSH-sepharose
itself, especially if you take in account the time and effort around
it and the starting materials. Of course, it might be an interesting
"hacking" experiment for an organic class, especially if you don't
need the material immediately and can live with some failures.

Just some thoughts for a start (I have no ready solution at hands):
The GSH is bound via the -SH group, you'll need a matrix that will
react with -SH, as eg maleimide activated resin. I guess you might
either find some info by checking the Methods in Enzymology series or
if you're lucky, someone has published a procedure you may get from a
paper to be found via http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search
or a patent like http://www.freepatentsonline.com

A possible synthetic path (I am just brainstorming) is to quench any
aminoreactive resin (eg NHS or CNBr activated sepharose) with a
several (say: 10) fold excess of a bis-amine like DADOO or 1,6-
diaminohexane an then convert the attached amino function to the
maleimide by reaction with possibly maleic anhydride or so (maybe
maleic acid + activator like DCC / NHS + base (DIPEA), you may adapt a
standard procedure for maleimides from an organic student course).
Then wash excessively and couple GSH and block with BME. If you need
longer spacers, attach e-aminocaproic acid first, activate with CDI or
carbodiimide/NHS and add the bis-amine then before making the
maleimide.

If you like to play with chemistry, you may cook something alike to
GSH with an amino residue at the thioether and stick it to CNBr- or
NHS activated sepharose, but that needs some protection/deprotection
(BOC, TFA) chemistry as there is another amino group in GSH you may
want to protect temporarily.

Another possible way could be to attach a molecule to the resin that
has a SH reactive group similar to NBD-Cl or NBD-F (check eg http://sial.com
for structures, these reagents are used to estimate SH residues in
proteins).

HTH
Wo

PS - any chemists out there? I'd appreciate your comments on my
proposals! Feasible? Something simpler available?


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