I wanted to screen for an antibiotic binding protein (aminoglycoside phosphotransferase). So the idea is to run a native PAGE of the crude protein extract and then incubate it with the antibiotic labelled with a fluorophore so that it binds with the respective protein and the specific band would fluoresce. I want to know whether this is possible or not and if yes how do we cross link it with the antibiotic?
Hi Vikas,
Yes I think it is possible. Since you have an amine group on the sugar, choose a N-hydroxysuccinimide ester labelled fluorophore. For the native PAGE, it could be a mess, so to improve you chances, check that the pI of other expected aminoglycoside phosphotransferases is below ~8.
Hi Vikas,
I agree with Carol. Just one advice, keep the molar ratio of kanamycin to N-hydroxysuccinimide ester labelled fluorophore 1:1. You don't want to decorate your kanamycin. It may lose the activity.
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