I am trying to inhibit the phosphorylation of a Erk 1/2 but the cells I am working with naturally have it already phosphorylated.
I want to see if I can remove the phosphate group with a specific phosphatase.
Try liposomes loaded with alkaline phosphatase. They will be taken up by living cells.
However, the chance that alkaline phosphatase will do its duty in the cell is doubtful. Acid phosphatase will destruct (lysosomal enzyme). Alkaline phosphatase is still active at pH 7.6
Do you want to dephosphorylate in the cells or after lysing the cells? Phosphatases are generally not very specific, so if you use an exogenous phosphatase in the living cells (e.g. by transient transfection of an expression plasmid or as suggested by Enrico) you might get all sorts of dephosphorylation events. This could seriously interfere with your experiment, even if you use the ERK phosphatases PTP or MKP1.
As an alternative you could try to block the phosphorylation of the ERKs by using a MEK specific kinase inhibitor (like e.g. AZD6244) prior to harvesting the cells which should reduce the phosphorylation level of the ERKs significantly.
If you just want to dephosphorylate ERK after cell lysis in-vitro you can use CIP which should work efficiently.
Hi, I'm not sure the context of your experiment , but if you must be specific to ERK 1/2, you may consider combining siRNA to all ERK and re-introduction of siRNA -resistant wt and phosphomutant ERK back into the cells. Good luck!
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