immune at intergate.bc.ca
Tue Mar 30 14:27:42 EST 1999
Iodoacetamide is a cysteine protease inhibitor as it react readily with
free thiol of the cysteine residues,which is required for the
hydrolytic activity of the proteases such as ICE, CPP32, Papain. The
Iodo group is most sensitive at pH.7-8 to the -SH, less extent (>2000
times) to -NH2 and -NH- and can also be rapidly inactivated by water if
pH is higher than 10. It is unlikely to cross-link as its amide end is
not very reactives. The NHS-iodoacetate ester is cross-linking agent
though. The addition of iodoacetate to the protein may slidely change
in molecular weight unless your protein is highly cysteine-rich. If you
are not sure that your protein is dimerized, and is not resolved by
SDS-PAGE in the presence of mercaptoethanol, then you have to do a
western blot to see if that protein is really your protein of interest.
In article <36FFD824.1C95 at maple.biology.yale.edu>,
claus at MAPLE.BIOLOGY.YALE.EDU says...
>I have recently had problems with my FPLC gel filtrations using yeast
>extracts. In my dispair, I added every single available protease
>inhibitor to my extract. Now I have a beautiful result but the protein
>comes out with approximately double the size and also runs according to
>doubled size on the gel. So, either I have modified my protein or
>cross-linked it with itself or another protein or ...
>The inhibitor I suspect having caused this is iodoacetamide. Can anyone
>tell me what the effects of iodoacetamide on proteins are?
>Thank you very much for your help.
>Please reply to Claus.Schwechheimer at yale.edu
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