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Kmu

Member Since 12 Sep 2012
Offline Last Active Mar 13 2013 04:45 PM

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Biochemistry and biophysics

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metionina
13 Sep 2012 - 03:45
ascacioc
12 Sep 2012 - 14:20

Topics I've Started

Cleavage of GST-tag with Factor Xa

13 March 2013 - 04:49 PM

Hello,

My GST-tagged protein has a Factor Xa site between the tag and the protein. In order to optimise conditions I have tried incubating 0.5U to 10U of Factor Xa with 5uM of my protein for time periods of 2-16 hrs. But when I run the samples on SDS-PAGE, I do not see anything. The gel looks like there was absolutely no protein loaded. I am unsure as to what is going on. Please help.
Thanks.

metal chelation for proteins

17 October 2012 - 06:22 PM

I am dealing with a metallo-enzyme. The idea is to chelate out the metals in it and try to replace it with other divalent metals to see if that improves activity. It is known that the protein needs 2 equivalents of metal to be catalytically most efficient. I have been growing the cells by adding the chelator at OD 0.2-0.3 and then adding the metal and IPTG at around OD 0.6. I am however, unable to ensure an uptake of 2 equivalents of metal by the protein (as evident by ICP-MS data) after an 18 hour growth. Is there any specific protocol that I need to follow to rectify my problem? Thanks.

how do GST-tags affect gel filtration results?

12 September 2012 - 08:23 AM

I am working with an insoluble protein which is associated with a GST-tag. The tag not only helps in keeping it soluble but also helps in purification. I am however, interested to know if the protein forms higher homo-oligomers. But I was reading about how running a GST-protein though a gel filtration column results in GST-GST interaction. This will not give me the results I wanted to look for. Also, cleaving the tag off makes the protein precipitate out. Please help.