2 replies to this topic

[biochem_student]

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?

[sve02594]

Hi:

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

Edited by sve02594, 18 September 2004 - 01:59 PM.

[biochem_student]

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.