Hello, I am using Promega's TNT in vitro coupled transcription/translation system with radio labelled 35S methionine and getting low to no yield with some of my proteins that I'm trying to express. I'm wondering if anyone has tried radio labelling with two amino acids i.e. methionine and cysteine, and if this will increase the amount of translated protein visible by x-ray film. I imagine that the more methionines + cysteines in my protein the greater the expression seen?
Thanks,
Becky
Just increasing the amount of radiolabeled amino acid may increase the yield of protein only if the total amount of the amino acid (hot + cold) is too small to support maximal translation.
If your protein contains no Met residues, then you will not see any product even if it is made because the product will not be radioactive.
Adding radiolabeled Met and Cys amino acids instead of just Met will increase the specific radioactivity of the product only if the product contains Cys residues, and therefore make it easier to detect by autoradiography or fluorography, as long as the total amino acid concentration is not decreased to the point that translation is decreased.
Another way to improve detection would be to increase the proportion of radiolabeled methionine to cold methionine, without changing the total amount of methionine.
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