Sep 23, 2023

Public workspaceIn vitro phosphatase assay

DOI
dx.doi.org/10.17504/protocols.io.ewov1qyepgr2/v1
  • 1Sascha Martens lab, University of Vienna, Max Perutz Labs - Vienna (AT)
Elias Adriaenssens
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DOI: dx.doi.org/10.17504/protocols.io.ewov1qyepgr2/v1
Protocol Citation: Elias Adriaenssens 2023. In vitro phosphatase assay. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov1qyepgr2/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 27, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 84093
Keywords: In vitro phosphatase assay, ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson’s (ASAP)
Grant ID: ASAP-000350
Marie Skłodowska-Curie MSCA Postdoctoral fellowship
Grant ID: 101062916
Abstract
This protocol describes in vitro phosphatase assay.
Attachments
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756-1924.pdf
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Materials
Materials

  • 6 well plates
  • MnCl2

ReagentPierce™ Detergent Compatible Bradford Assay KitThermo FisherCatalog #23246

ReagentLambda Protein Phosphatase - 100,000 unitsNew England BiolabsCatalog #P0753L

Lysis buffer
AB
HEPES pH 7.450 mM
NaCl150 mM
MgCl22.5 mM
DTT2 mM
NP-400.50%
protease inhibitor cocktail
In vitro phosphatase assay
In vitro phosphatase assay
5m
5m
Seed HAP1 wild-type or FIP200 knockout cells in 6 well plates and grow until confluency.
Collect cells by trypsinization and pellet by centrifugation at Centrifigation300 x g, 4°C, 00:05:00 .

5m
Centrifigation
After a PBS wash to remove the remaining cell medium, resuspend the cell pellets in lysis buffer.
Wash
Lyse the samples for Duration00:20:00 TemperatureOn ice . Clear the cell lysates by centrifugation at Centrifigation20000 x g, 4°C, 00:10:00 .

30m
Centrifigation
Digestion
Determine the protein concentrations of the cleared protein lysates with the Pierce Detergent Compatible Bradford Assay Kit (23246, Thermo Fisher).
For both samples, wild-type and FIP200 knockout lysates, incubate Amount100 µg of cell lysate with Amount5 µL of 10x NEBuffer for Protein MetalloPhosphatases (P0753, New England Biolabs) and Amount5 µL of Concentration10 millimolar (mM) of MnCl2 to make a total reaction volume of Amount50 µL .
Incubation
Add Amount1 µL of Lambda Protein Phosphatase (NEB) to the reaction and incubate the samples at Temperature30 °C for the indicated time.
Incubation
Pipetting
Terminate the phosphatase reactions by the addition of 6x Protein Loading dye and heat inactivation at Temperature95 °C for Duration00:05:00 .
5m
Temperature
Analyze the samples by western blot analysis as described above.
Analyze
Protocol references