Protein Phosphatase Assay

Materials
	black flat-bottom 384-well plates (Greiner Bio-One, Germany)
	phosphatase buffer (50 mM TrisHCl, pH = 8.0 at 25°C, 200 mM NaCl, 20 mM MnCl2, 2 mM TCEP, 0.01% Tween). TCEP and Mn2+ are added freshly; 1L bottle is sterile filtered and stored at 4°C.
	4-Methylumbelliferyl phosphate (4-MUP) (Sigma Aldrich, Germany) or DiFMUP (ABCR GmbH) is dissolved in DMSO (10 mM stock solution) and stored at -50°C
	recombinantly expressed phosphatase (-80°C)
	compound aliquots (-80°C) and DMSO for pre-dilution

Assay Procedure

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1.)  Prepare buffer with and without Mn²⁺ in appropriate amount

2.)  Pre-dilute substances in DMSO in a 96 deep-well plate (can be frozen at - 80°C)

Endkonzentration im Assay [µM]	Total- Faktor	Verdünnung in DMSO [µM]
10	X1000	10000	→	0	µl DMSO	+	5	µl aus	10mM
3,3	X1000	3333	→	6	µl DMSO	+	3	µl aus	10
1	X1000	1000	→	7	µl DMSO	+	3	µl aus	3333
0,3	X1000	333	→	6	µl DMSO	+	3	µl aus	1000
0,1	X1000	100	→	7	µl DMSO	+	3	µl aus	333
0,03	X1000	33	→	6	µl DMSO	+	3	µl aus	100
0,01	X1000	3	→	7	µl DMSO	+	3	µl aus	33
0	X1000	0	→	7	µl DMSO

3.)  Pre-dilute DMSO dilution row again in assay buffer (125 µl buffer and 0.5 µl DMSO dilution) in another 96 deep-well plate to result in 1:250 dilution (in the assay again 1:4 dilution is done to result in a total dilution of 1:1000).

4.)  Freshly prepare phosphatase enzyme solution using appropriate amounts of phosphatase. PC = phosphatase with Mn²⁺ buffer (60µl per well); NC = phosphatase w/o Mn²⁺ (60µl per well); samples = phosphatase with Mn^2+-buffer (40µl per well). Freshly prepare DiFMUP/4-MUP solution by adding appropriate amounts to assay buffer w/o Mn²⁺.

5.)  Add 80µl of one buffer in the first and the last column of a 384-well plate (not measured because of effects in the reader); add 60 µl/well (controls) or 40 µl/well (samples) of phosphatase solution for controls and samples; add 60µl/well of buffer with and w/o Mn²⁺ for background detection during the assay into wells (Blanks = BL1 and BL2).

6.)   Add 20 µl of different compound solutions (10µM - 0µM) to the sample wells, mix and pre-incubate for 10 min at 35°C under constant shaking (5 Hz).

7.)   For starting the reaction add 20 µl of the substrate solution to all wells yielding a final concentration of 100 µM DiFMUP/4-MUP.

8.)   Fluorescence intensities of the hydrolyzed 4-Methylumbelliferone (4-MU) or DiFMU (excitation 360nm/emission 448nm) are monitored by a microplate reader (Varioscan, Thermo Fisher Scientific) in a kinetic mode every 5 min over a 60 min period at 35°C.

9.)   For calculation of final values normalize to auto-hydrolysis (blanks) and autofluorescence of compounds (first time-point). You can draw curves with GraphPadPrism and align IC50/EC50 curves or use end-point values etc.

Example for a 384-well plate: In color - Compound dilution rows in quartets for 6 compounds