xgpt activity

Christine Gee gee at florey.biosci.uq.oz.au
Tue Sep 21 21:45:49 EST 1993


Mats.Nilsson at bio.embnet.se (Mats Nilsson) writes:

>Hello bionetters:

>I been working with a plasmid construction including xgpt as a selectable
>marker, with this we produce RNA to transfect COS cells. Now the problem is
>the lack of a reliable xgpt assay.
>I wonder if anyone can tell me a good and "simple" method for detection of
>xgpt activity (xgpt=xanthine guanine phosphoribosyltransferase from E
>Coli).
>I would apreciate any references and suggestions.

>Thanks a lot, 
Dear Jorge
We use a spectrophotomertic assay.In our lab, using  1 ml of tris buffer pH 8.5
(.1 M) with 0.11M MgCl2) 30 uL guanine (20 mg/50mL) and 50 uL PRPP (20 mg/ml)
and measure the increase in absorbance at 257.5 nm in a ml quartz cuvette.
To this up to 100 ul of enzyme can be added. the reaction is incubated at 25 C
or 37 C depending on you (I assay HPRT at 25 but my fellow PHD student assays
XPRT expressed in e.coli at 37)
The guanine solution needs to be at about pH 12 before it will dissolve. PRPP
is unstable and should be frozen in solution. guanine can be stored in the 
fridge.  There is also a radiochemical assay. If you are interseted in this
send me an email. Christine (gee at florey.biosci.uq.oz.au) 
.



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