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Malachite green phosphate assay

RB Rebecca Brinkschulte
DF David M. Fußhöller
FH Florian Hoss
JR Juan F. Rodríguez-Alcázar
ML Mario A. Lauterbach
CK Carl-Christian Kolbe
MR Melanie Rauen
SI Semra Ince
CH Christian Herrmann
EL Eicke Latz
MG Matthias Geyer
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The malachite green phosphate assay kit (Cayman Chemical, Ann Arbor, USA) was used to measure the amount of inorganic phosphate (Pi) generated during an ATP hydrolysis reaction, according to the manufacturer’s instructions. The proteins to be tested were diluted to a final concentration of 0.4 µM using a 10x buffer containing 200 mM HEPES pH 8.0, 1.5 M NaCl, 50 mM MgCl2, and incubated with ATP (Jena Bioscience, Jena, Germany) for 30 min (25 °C) or over a time course of 240 min (20 °C). Absorbance at 620 nm was measured using a Tecan plate reader (Tecan Trading AG, Männedorf, Switzerland). The standard curve and the protein samples were measured in technical duplicates.

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