. Author manuscript; available in PMC: 2011 Oct 1.

Published in final edited form as: Curr Protoc Mol Biol. 2010 Oct;CHAPTER:Unit18.18. doi: 10.1002/0471142727.mb1818s92

Table 3.

Commonly encountered problems.

Problem	Possible Cause	Solution

High Background (high signal with no added phosphatase)	Interfering substances present (malachite green assay)	Remove trace amounts of phosphate from assay buffers/reagents
	Insufficient removal of unincorporated label (³²P- labeled substrates)	Perform extra desalting or dialysis step to remove unincorporated label
	Protease contamination (³²P- labeled substrates)	Add protease inhibitors to prevent substrate hydrolysis
	Phosphatase contamination of substrate stock solution	Prepare new substrate
	Substrate has denatured or aggregated (³²P-labeled substrates)	Ensure proper storage conditions. Prepare new substrate using new lot of protein

Expected phosphatase activity is not detected	Phosphatase is missing or inactivated	Use a positive control phosphatase or standard curve to check assay
	Activators not present	Examine reaction mix for necessary cofactors

Cloudy precipitate (malachite green assay)	Interfering substances present	Identify and remove incompatible metals, phosphate, detergents

Weak signal	Phosphatase too dilute	Increase amount of phosphatase or increase the assay time