purification of RNAP from E.coli
Posted 18 September 2004 - 10:36 AM
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?
Posted 18 September 2004 - 01:59 PM
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.
Posted 18 September 2004 - 02:29 PM
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.