hej, I am having troubles with something that is supposed to be quite trivial, so I am most likely doing something stupid and would be very happ if somebody tells me what is it : )
So, I dissolve 100um L-cysteine in 150mM NaCl and I add 10uL of 100mM iodoacetamide. I incubate this for cca 1hour in the dark and then I assay free thiols. ... and it doesn't work, I still get redox activity. I tried increasing iodoacetamide concentration to 5mM, no success.
//Iodoacetamide is bought new, freshly prepared and kept in the dark. //
So ... what I am doing wrong?!
Thanks a lot!
It may that your solution is too acid for the reaction. Reaction of iodoacetamide with -SH from cysteines works better at slightly alkaline pHs.
"Perform alkylation with limiting quantities of iodoacetamide at a slightly alkaline pH (pH8-9) to ensure alkylation is exclusive to cysteine residues. Excess or non- buffered iodoacetamide may result in alkylation of lysines, N-termini, methionines, histidines, aspartates and glutamates. The supplied alkylation buffer should be added to the solutions to be alkylated to ensure exclusive cysteine residue alkylation. "
From http://www.gbiosciences.com/PDF/Protocol/FOCUS_Protein_Alkylation.pdf
Hope this helps
Brandán is right, you may be using Cys chlorhydrate as starting reagent wich renders your pH acidic. The reaction is too slow under these conditions, try at neutral or alkaline pH. All the best, GFS
Thank you very much!
I actually kept my solution at around pH 7.0 but found out that reaction works if I put the solution for half an hour on 37°C.
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