Sep 23, 2023

In vitro phosphatase assay

DOI
https://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: https://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, phosphatase assay, phosphatase, assay this protocol, assay
Funders Acknowledgements:
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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Materials
Materials

  • 6 well plates
  • MnCl2

Pierce™ Detergent Compatible Bradford Assay KitThermo FisherCatalog #23246

Lambda 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
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 300 x g, 4°C, 00:05:00 .

5m
After a PBS wash to remove the remaining cell medium, resuspend the cell pellets in lysis buffer.
Lyse the samples for 00:20:00 On ice . Clear the cell lysates by centrifugation at 20000 x g, 4°C, 00:10:00 .

30m
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 100 µg of cell lysate with 5 µL of 10x NEBuffer for Protein MetalloPhosphatases (P0753, New England Biolabs) and 5 µL of 10 millimolar (mM) of MnCl2 to make a total reaction volume of 50 µL .
Add 1 µL of Lambda Protein Phosphatase (NEB) to the reaction and incubate the samples at 30 °C for the indicated time.
Terminate the phosphatase reactions by the addition of 6x Protein Loading dye and heat inactivation at 95 °C for 00:05:00 .
5m
Analyze the samples by western blot analysis as described above.
Protocol references