TITLE: Assay for quantifying PPM1H phosphatase activity towards LRRK2 phosphorylated Rab proteins and peptides using the Malachite Green method
AUTHORS: Kerryn Berndsen, Dario Alessi

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">We describe a sensitive and non-radioactive assay that we deploy for analysing PPM1H protein phosphatase activity using either Rab8A stoichiometrically phosphorylated at Thr72 (the LRRK2 site) or a phospho-peptide encompassing this residue. Our assay relies on malachite green, an inexpensive basic dye, that in the presence of molybdate forms a green complex with orthophosphate hydrolysed from the phospho-substrate during the PPM1H phosphatase reaction. This green malachite orthophosphate complex can be conveniently measured on a spectrophotometer (620 nm). The assay is straightforward and rapid. It involves a single addition step for initiating and terminating the reaction. The assays can be conveniently performed in 96 well plate format and can be adapted to compare the intrinsic activity of different protein phosphatases as well as impact of mutations.</div></div>

[STEPS]
?. [Assay for quantifying PPM1H phosphatase activity towards LRRK2 phosphorylated Rab proteins and peptides using the Malachite Green method]
Setup the assays in a 96-well flat-bottomed plate (see Figure 1, Figure 2B and Figure 3B):
?. [Assay for quantifying PPM1H phosphatase activity towards LRRK2 phosphorylated Rab proteins and peptides using the Malachite Green method]
Label the plate with the appropriate time points above each well to ensure no mix-up.
?. [Assay for quantifying PPM1H phosphatase activity towards LRRK2 phosphorylated Rab proteins and peptides using the Malachite Green method]
Initiate the phosphatase assay reaction by briefly pausing the orbital shaker and adding of phosphatase mixture to the desired wells of the 96-well plate for each timepoint.
5 µl
?. [Assay for quantifying PPM1H phosphatase activity towards LRRK2 phosphorylated Rab proteins and peptides using the Malachite Green method]
Add  of required phospho-substrate to a sufficient number of wells required to perform the planned number of assays.
75 µl
We recommend undertaking each assay condition in at least duplicate, preferably in triplicate.
?. [Assay for quantifying PPM1H phosphatase activity towards LRRK2 phosphorylated Rab proteins and peptides using the Malachite Green method]
Keep plate and aliquoted samples at .
0 Room temperature
?. [Assay for quantifying PPM1H phosphatase activity towards LRRK2 phosphorylated Rab proteins and peptides using the Malachite Green method]
Add  of the serial dilution of orthophosphate standards to another set of wells in duplicate.
80 µl
?. [Assay for quantifying PPM1H phosphatase activity towards LRRK2 phosphorylated Rab proteins and peptides using the Malachite Green method]
Place the 96-well plate at room temperature on an orbital shaker with slight agitation.
?. [Assay for quantifying PPM1H phosphatase activity towards LRRK2 phosphorylated Rab proteins and peptides using the Malachite Green method]
Mix well by pipetting up and down before restarting the orbital shaker.
If a time course study is being undertaken, we recommend initiating the assay with the longest timepoint first and working backwards so that all assays are terminated at a similar time.
?. [Assay for quantifying PPM1H phosphatase activity towards LRRK2 phosphorylated Rab proteins and peptides using the Malachite Green method]
When the desired time point to terminate the reaction is reached, stop reactions by adding of malachite green reagent to each phosphatase reaction and mixing by pipetting up and down.
20 µl
The malachite green reagent is acidic and will denature the phosphatase and therefore stop the reaction.
?. [Assay for quantifying PPM1H phosphatase activity towards LRRK2 phosphorylated Rab proteins and peptides using the Malachite Green method]
For the ‘zero’ timepoint add of the malachite green reagent to  of the phospho-substrate mixture followed by of the phosphatase mixture.
20 µl
75 µl
5 µl
?. [Assay for quantifying PPM1H phosphatase activity towards LRRK2 phosphorylated Rab proteins and peptides using the Malachite Green method]
Incubate the 96-well plate at  for  to allow the green colour to develop.
0 Room temperature
?. [Assay for quantifying PPM1H phosphatase activity towards LRRK2 phosphorylated Rab proteins and peptides using the Malachite Green method]
Measure the absorbance at 620 nm using spectrophotometric multi-well plate reader.
?. [Assay for quantifying PPM1H phosphatase activity towards LRRK2 phosphorylated Rab proteins and peptides using the Malachite Green method]
Plot absorbance (620nm) versus phosphate standard concentrations to create a standard curve.
?. [Assay for quantifying PPM1H phosphatase activity towards LRRK2 phosphorylated Rab proteins and peptides using the Malachite Green method]
Determine concentration of phosphate released for each assay using the standard curve.
?. [Assay for quantifying PPM1H phosphatase activity towards LRRK2 phosphorylated Rab proteins and peptides using the Malachite Green method]
Subtract the zero timepoint from concentration of phosphate released from timecourse.
?. [Assay for quantifying PPM1H phosphatase activity towards LRRK2 phosphorylated Rab proteins and peptides using the Malachite Green method]
Plot the amount of phosphate released (μM) vs time (min) (See Figure 2A and Figure 3A that shows a time course of PPM1H dephosphorylation of  stoichiometrically Thr72 phosphorylated Rab8A.
These assays also demonstrate that catalytically inactive PPM1H[D288A] mutant fails to dephosphorylate the phospho-Rab8A protein or phopsho-Rab8A peptide. The assays also demonstrate that PPM1M and PPM1J phosphatases that are closely related to PPM1H poorly dephosphorylate the phosphoRab8A protein and peptide substrate).