TNAP Malachite Green Assay (384-well)
TNAP’s typical substrates are inorganic pyrophosphate (PPi) and pyridoxal-5′-phosphate (PLP) as well as probably phosphorylated osteopontin (p-OPN), and adenosine tri-/di-/mono-phosphate (ATP/ADP/AMP).
The Malachite Green Assay is based on quantification of the green complex formed between Malachite Green, molybdate, and free orthophosphate (Pi). The rapid color formation from the reaction can be conveniently measured absorbance at 600–660 nm (620 nm).
Assay Procedures
1.Echo dispense 20 nL serial diluted compounds to 384w assay plate.
2.Add 10 μL of 0.1 ng/μL rhTNAP to wells (FAC 0.05 ng/μL).
3.Incubate at RT for 10 min.
4.Add 10 μL of 60 μM AMP substrate to wells (FAC 30 μM).
5.Incubate at RT for 30 min.
6.Stop reaction by adding 20 μL of the malachite green color reagent [0.03% (w/v) malachite green oxalate, 0.2% (w/v) sodium molybdate, 0.05% (v/v) Triton X-100, dissolved in 0.7 M HCl]
7.Incubate at RT for 30 min.
8.Quantify inorganic phosphate (Pi) by measuring OD620
Result: TNAP inhibitor SBI-425 IC50 was 45.3 nM
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