Here activity is measured by incubating the protein with a substrate and reading the value at appropriate wave length as the reaction is expected to release a fluorescent product.
The activity is very weak compared to many related phosphatases, so could not use regular methods like pNPP or malachite green assay. Inhibition of this weak activity by the phosphatase inhibitor Na3VO4 makes me wonder if the protein is a phosphatase? I appreciate your thoughts on this. Have you come across any such examples?
The sodium orthovanadate (Na3VO4) is a commonly used general inhibitor for protein tyrosine phosphatases (PTPs), alkaline phosphatase (ALP) and ATPase. This acts as a competitive inhibitor and is completely reversible upon the addition of EDTA or by dilution, because the interaction is not covalent type. Look at this paper, is really good.
http://www.sciencedirect.com/science/article/pii/0076687991010432
But to be more certain if your protein is really a phosphatase, using malachite green assay in a sample with EDTA, your protein and Na3VO4 (must be obtained fluorescence). Besides, do the same assay but without EDTA and vanadate, so confirm that it is not a problem with the substrate and the protein.
Thank you so much Carlos. Malachite green assay doesn't work. pNPP assay doesnt work but Difmup assay works.....Thanks for the reference.
Thanks Kuppusamy. Have you worked on weak phosphatases before? Just curious.
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