Phosphatase Assay help - (Nov/07/2006 )
Can anyone help me with a simple to use phosphatase assay protocol where I don't have to buy a kit.
I am hoping to look at the phosphatase activity of a phosphatase pulled out of a mammalian cell lysate by IP. This phosphatase has a known substrate (P-ERK) and I was thinking of possibly IP'ing T-ERK from cells treated with EGF to act as a substrate and then mixing them together in a compatible buffer and then monitoring P-ERK levels by WB.
I know there are many possible problems with this idea such as,
Ab binding may interfere with the active site/dephopsphorylation site
Additional proteins may co-IP with the enzymes/substrates
The stoichoimetry of the reaction may not be right
To name but a few
However its one of those experiments I want to do "under the bench" so cannot really order anything for the time being.
Has anyone tried to look at phosphatase activity in a similar way? And does anyone have a phosphatase assay compatible buffer?
Thanks in advance
I am hoping to look at the phosphatase activity of a phosphatase pulled out of a mammalian cell lysate by IP. This phosphatase has a known substrate (P-ERK) and I was thinking of possibly IP'ing T-ERK from cells treated with EGF to act as a substrate and then mixing them together in a compatible buffer and then monitoring P-ERK levels by WB.
I know there are many possible problems with this idea such as,
Ab binding may interfere with the active site/dephopsphorylation site
Additional proteins may co-IP with the enzymes/substrates
The stoichoimetry of the reaction may not be right
To name but a few
However its one of those experiments I want to do "under the bench" so cannot really order anything for the time being.
Has anyone tried to look at phosphatase activity in a similar way? And does anyone have a phosphatase assay compatible buffer?
Thanks in advance
what you need is an idea of your PPP; your PPP against P-ERK, is it a Y- or a T/S-PPP? even with IP you may co-precipitate various PPP; so to address a specific PPP, you may try various specific PPP inhbitiors; global inhibitors for PPP such as fluoride, naphtyl-phosphate, vanadate and so on, are not to recommend; if you know your PPP, there is perhaps a specific inhibitor available;