Table 18.16.2.

Troubleshooting PTPase Assays

Problem	Solution
pNPP assay and Malachite Green assay
Colorimetric response too weak	Use more cells for immunoprecipitation
	Incubate PTP and beads for longer time
	Use a positive control (another PTP) to check reagent quality
Colorimetric response too strong	Use fewer cells for immunoprecipitation
	Incubate PTP and beads for shorter time
	Use a negative control to check background
Radioactive in-gel phosphatase assay
No white bands visible (all black)	Renaturation ineffective, use fresh DTT
	Use shorter exposure
Too light background	Control in-vitro kinase conditions
	Use fresh γ32P
	Use longer exposures
In-vitro and in-cell substrate-trapping
No tyrosine-phosphorylated protein detected	Run total cell lysate to check for overall protein tyrosine phosphorylation (should be increased in vanadate-treated or stimulated cells)