ESO wikiadmin: Created page with "{| class="wikitable" |+ !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 |- | |rec..."

2025-02-27T12:06:05Z

Created page with "{| class="wikitable" |+ !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 |- | |rec..."

New page

{| class="wikitable"
|+
!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 ===
----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)
{| class="wikitable"
|Endkonzentration im Assay [µM]
|Total- Faktor
|Verdünnung in DMSO [µM]
|
|
|
|
|
|
|
|-
|10
|X1000
|10000
|→
|0
|µl DMSO
|<nowiki>+</nowiki>
|5
|µl aus
|10mM
|-
|3,3
|X1000
|3333
|→
|6
|µl DMSO
|<nowiki>+</nowiki>
|3
|µl aus
|10
|-
|1
|X1000
|1000
|→
|7
|µl DMSO
|<nowiki>+</nowiki>
|3
|µl aus
|3333
|-
|0,3
|X1000
|333
|→
|6
|µl DMSO
|<nowiki>+</nowiki>
|3
|µl aus
|1000
|-
|0,1
|X1000
|100
|→
|7
|µl DMSO
|<nowiki>+</nowiki>
|3
|µl aus
|333
|-
|0,03
|X1000
|33
|→
|6
|µl DMSO
|<nowiki>+</nowiki>
|3
|µl aus
|100
|-
|0,01
|X1000
|3
|→
|7
|µl DMSO
|<nowiki>+</nowiki>
|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 Mn2+-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.
[[File:Example384wellplate.png|thumb|Example for a 384-well plate: In color - Compound dilution rows in quartets for 6 compounds]]

Protein Phosphatase Assay - Revision history