Does anyone know of an assay for alkaline phosphatase that is more
sensitive than the colorimetric (i.e. p-nitrophenyl phosphate) one? A
radiochemical one, perhaps? or could I adapt one of the chemiluminescent
southern/northern protocols? A problem I see here is that the
luminescent products are (necessarily) insoluble wheras for an enzyme
assay it would be better if it were soluble...
Alternatively, perhaps the molecular biologists among you can help
me....
I am trying to determine the topology of a multi-membrane spanning
protein which is pretty toxic, even in small segments. I have made a
series of phoA fusions in a low/medium copy number vector. These are
stable, but the alkaline phosphatase activity is too low to be useful. I
have tried transferring the some of the constructs to a high copy number
vector, but the colonies, while nicely blue in parts, develop white
sectors suggesting deletion of the inserts. How should I proceed? Should
I clone the inserts under the control of a regulated promoter and induce
when I am ready to do the measurements?
Any ideas gratefully received. Thanks.
Dudley Page (mpage at worf.molbiol.ox.ac.uk)
