dhfr activity assay?

Steven Tait STAIT at lab0.vet.ed.ac.uk
Sun Aug 11 08:09:33 EST 1996


Shanthi Adimoolam <shanth at students.uiuc.edu> wrote:
>
> Hi,
> I am trying to overproduce some mutants in mammalian cells which are 
> deficient in their normal activity. The selectable marker that i am using is the dhfr gene 
> and i am expressing these mutants in CHO/dhfr- cells. I am wondering if 
> there is an assay for dhfr (dihydrofolate reductase) that will enable me 
> to select for colonies expressing high levels of the mutant protein. Any 
> reference or some info would be extremely helpful.
> Thanks in advance.
> -shanthi
> 
Hi
To detect DHFR activity from bacteria on IEF gels I used to use an 
MTT based assay. The MTT changes colour in a reduced environment eg 
when DHF is reduced to THF in NADPH linked DHFR catalysed reaction.
So your assay may be possible but it will take some doing, and a lot of tweaking.
I can't remember the details off hand but I will see if I can dig out the
assay conditions I used.

Steven




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