I am looking for the S-Carboxymethylation of Cysteine protocol. I want to methylate my protein by using iodoacetic acid. If you have a procedure for that, please send my a copy or tell me where I can reach it.
Thank you in advance
Look for the protocol in "www.ionsource.com". Alkylation of Cysteine thiols: By using 50 or 100 mM ammonium bicarbonate, maintain the peptide or protein sample at pH 7.5-8.0. Add 50 or 100 mM iodoacetamide (IAM) or iodoacetic acid (IAA) and incubate in dark (since IAA or IAM is light-sensitive) at room temperature (27-30 degree Celsius) for about 45 mins. - 1 hour. And then the sample can be characterized by mass spectrometry. From the increase in the molecular mass, the number of cysteines can be known. This protocol is useful for determining free cysteines (thiols) in the peptide/protein. The concentration mentioned above are only representative. It needs to be optimized for different protein/peptide samples, depending on their concentrations. Reduction reaction of Disulfide bonds: In case, the protein/peptide contains 'disulfide bonds', then prior to IAM/IAA treatment, the disulfide bonds need to be reduced. For reduction, the protein/peptide need to be treated with Dithio threitol (DTT) or beta-mercaptoethanol at pH 7.5-8.0, using ammonium bicarbonate. The reduction reaction can be done at 37or 50 or 60 degree Celsius. Depending on the temperature followed and depending on the protein/polypeptide, the time of incubation can be varied from 30 mins. - 3 hours. Subsequently, the reaction mixture must be brought to room temperature and then the alkylation reaction (i.e. treatment with IAA or IAM) needs to be performed. Overall, this is called as 'Reduction-Alkylation reaction of Disulfide bonded polypeptides/proteins.
Hello Dr.Sabareesh,
Do you have any idea to how get rid of Iodoacetic acid after alkylation reaction? I am planning to do in-vitro protein-protein interaction and the IAA might affect the binding reaction. By the way, I have a limited amount of a protein that I will alkylate.
Thanks in advance
You may try using either membrane filters of 3 kDa Molecular Weight Cut-Off (MWCO) or HPLC (reverse phase) with a suitable column, say C8 or C4, depending on your protein or Gel Filtration (Sephadex). In all these cases, you need to do experiments carefully, since you have limited quantity of sample.
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