Problem	Possible Cause	Solution
Low transfection efficiency	Different transfection reagent. Low/high cell confluency, insufficient plasmid DNA used for complex formation. Plasmid DNA is of poor quality or degraded.	Select suitable transfection reagent based on the cell type used for assay. Optimize transfection protocol by using different cell confluency, plasmid concentration and transfection duration. Re-purify plasmid and store in small aliquots at −20 °C.
No color appearance in pNPP reaction	Post-lysis oxidation occurred. Lysate stored on ice for long period Low protein input. Unsuccessful IP. Unsuccessful dephosphorylation of pNPP pNPP buffer was not degassed efficiently	Measure pO2 levels of lysis buffer with an oxygen electrode before using for cell lysis. Time to perform cell lysis, Bradford, IP and pNPP reaction should be optimized. Protein concentration should be optimized for your PTP of interest. Optimize duration, antibody and protein concentration for IP. Optimize duration of pNPP reaction or the concentration of substrate. Boil and degas ddH2O and verify pressure or leaks on vacuum pump
No band on western blot	Degassing of buffers. Protein degradation. Low protein input. Unsuccessful IP. Unsuccessful IAP labeling. Insufficient denaturation of sample in Laemmli buffer. Compromised epitope	Measure pO2 levels of lysis buffer with an oxygen electrode before using for cell lysis. Avoid keeping cell lysates on ice for a long period. Use fresh stocks of protease inhibitors. Protein concentration should be optimized for your PTP of interest. Optimize duration, antibody and protein concentration for IP. IAP should be made fresh. Post-lysis oxidation of PTPs and the pH of the lysis buffer can also affect IAP labeling. Heat samples for 90-120 seconds at 95 °C. Final concentration of sample buffer should be at least 1X. IAP binds to the catalytic cysteinyl residue of PTPs. Antibodies used to detect the PTP should not target an area close to the active site of the PTP.