IUBio

Endogenous phosphatase

Glen Tamura gtamura at u.washington.edu
Fri Jul 23 15:25:20 EST 1999


Paula:

Some organisms have more endogenous phosphatase than others. If you are
doing this in E. coli, there shouldn't be a huge problem with endogenous
phosphatases if you use a colorimetric substrate, as opposed to
chemilumiescence, which is often so sensitive that it gives whopping
signals with everything. 

You might also consider using an HRP-conjugated secondary antibody.
Peroxidase is produced by some microorganisms, but not others, so it might
prove to give you a lesser background. 

Good luck!

Glen Tamura

On Wed, 21 Jul 1999, Paula Murphy wrote:

> I am doing some plaque lifts and subsequently washing a nitrocellulose
> membrane in 0.5%milk-TBS buffer and secondary antibody conjugated with
> alkaline phosphatase. The method that I use to detect the bound secondary
> antibody also detects the endogenous phosphatase. If I add organic
> phosphate to the medium and wash the membranes with PBS instead of TBS
> will the production of alkaline phosphatase decrease?(My results are
> obtained on xray film)
> 
> --Thanks,
> paulam at ganymede.cs.mun.ca
> 
> =========================
> 
> 
> 
> 




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