Questions about protein quantification - from the industry perspective

Trond Erik Vee Aune via methods%40net.bio.net (by trondaun from REMOVE.nt.ntnu.no)
Tue Jun 3 01:54:49 EST 2008


WS skrev:
> Hi Trond,
> 
> what about beta-galactosidase or beta-glucuronidase? There are
> chromogenic and fluorogenic subtrates available.

Are you suggesting using the alpha subunit of beta-galactosidase as a 
reporter tag (analogous to blue/white screening)? If so I worry that the 
signal strength will be inhibited by low levels of the omega subunit 
(expressed from the chromosome). And I don't know if this will function 
at all in other bacteria except E. coli.

Another possibility is of course to use the whole beta-galactosidase as 
a reporter, and this is presumably what you meant, but unfortunately 
this protein is very large (1021 aa) and might therefore not be suitable 
as a reporter tag (?). And would the presence of chromosomal copy raise 
the background too high?

This last question is relevant for beta-glucuronidase too. 
Unfortunately, in addition, use of the GUS assay is covered by patents, 
so this might be an expensive alternative.

Trond Erik








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