Alkylation of cysteine residues

Ruediger Hornig hornig at bc.biol.ethz.ch
Fri Mar 7 10:38:41 EST 1997


Ulrik Sidenius <ulsi at charon.dfh.dk> wrote:
> I am looking for a method for reduction and alkylation of proteins
> before running SDS-PAGE.

Hi Ulrik

we do alkylation by adding a final concentration of 25 mM NEM
(N-ethyl-maleimide) to the boiled gel samples (reducing agent is 3 mM
DTT). This alkylates the DTT and all the cysteins in the protein. As an
alternative, you may alkylate the unreduced protein after purification to
block free sulfhydryls, dialyze excess of NEM out if needed and boil your
gel samples as usual.

hope this helps

Rudi

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