I have purified a transporter membrane protein and want to perform a CPM assay to assess its melting temperature as apo- and with ligands. The melt scan results in steadily dropping curve while my control protein melts fine (still a blue shift). My target melts with an orthogonal method (tryptophan DSF) but gives a broad transition and therefore wanted to try CPM as well.
My target protein has an abundance of cysteines while it seems the control has much less and has them located in TMs instead of solvent exposed loops (no wonder it worked for control and not for my target).
I thought about alkylating the solvent exposed cysteines with reagents like iodoacetamide and derivatives. Is this possible for an already purified membrane protein? Most protocol suggest to add to membranes before purification. What about side reactions? What is the best setup to selectively alkylate solvent exposed cysteines?
Interesting question. Looking forward to the discussion by fellow experts.
Interesting question. Looking forward to the discussion by fellow experts.
You can do the alkylation, but clean the protein again by gel filtration or dialysis. There are small ultrafiltration or dialysis cassettes available for such reactions.
I have labelled semi-purified Na/K-ATPase with IAEDANS (a fluorescent derivative of iodoacetamide), that worked fine. Just quench any excess with a thiol and then remove the low molecular mass compounds, for example with a desalting column.
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