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purification of RNAP from E.coli - (Sep/18/2004 )

I'm an undergrad biochemistry student. I have a few questions concerning the protocol for purifying RNAP from E.coli.

First of all, I'm not quite certain why dithiothreitol (DTT) is contained in the buffers to elute the proteins from a chromatographic column. And what is the purpose of glycerol in these buffers?

In assaying RNAP activity, I noticed the addition of B-mercaptoethanol in the assay mix. How does B-mercaptoethanol enhance enzyme stability?

Finally, with the fraction containing the enzyme. what is the purpose of adding glycerol to it for assaying the enzyme activity? ie 50% glycerol? My final absorption peak at 280nm is larger in the presence of glycerol than just the enzyme alone. what's up with that?

can somebody explain this?



If you can tell me more clearly how you are assaying RNAP activity....may be then I can answer your questions.


hi, thanks for your reply

This RNAP assay is based on the incorporation of labelled nucleotides (tritiated UTP) into RNA in the presence of a DNA template. The RNA chains are absorbed on a DEAE filter disc, while the unincorporated tritiated UTP are washed off. Okay, that's my assay.