Mar 21, 2025
In Vitro Phosphatase Assay
- 1Sascha Martens lab, University of Vienna, Max Perutz Labs - Vienna
Protocol Citation: Elias Adriaenssens 2025. In Vitro Phosphatase Assay. protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvm9b95l3p/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: February 10, 2025
Last Modified: March 21, 2025
Protocol Integer ID: 120012
Keywords: ASAPCRN
Funders Acknowledgements:
Aligning Science Across Parkinson’s (ASAP)
Grant ID: ASAP-000350
Marie Skłodowska-Curie MSCA Postdoctoral fellowship
Grant ID: 101062916
Abstract
This protocol details the in-vitro phosphatase assay.
Materials
Lysis Buffer:
| A | B | |
| Tris- HCl, pH 7.4 | 20 mM | |
| NaCl | 150 mM | |
| EDTA | 1 mM | |
| EGTA | 1 mM | |
| Triton X-100 | 1% | |
| Protease-inhibitor cocktail, but no phosphatase inhibitors | ||
1x phosphatase buffer:
| A | B | |
| Tris- HCl, pH 7.4 | 50 mM | |
| NaCl | 100 mM | |
| DTT | 2 mM | |
| MnCl2 | 1 mM |
- Pierce™ Detergent Compatible Bradford Assay KitThermo FisherCatalog #23246
Procedure
Procedure
1h 5m
1h 5m
To verify the activity of our recombinantly purified λ phosphatase, collect HeLa cells by trypsinisation and wash the cell pellet with PBS once before cells were lysed in lysis buffer.
Lysis buffer:
| A | B | |
| Tris- HCl, pH 7.4 | 20 mM | |
| NaCl | 150 mM | |
| EDTA | 1 mM | |
| EGTA | 1 mM | |
| Triton X-100 | 1% | |
| Protease-inhibitor cocktail, but no phosphatase inhibitors | ||
Lyse the cells for 00:20:00 On ice before cell lysates were cleared 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) and incubate the equal amounts with or without home-made λ phosphatase in 1x phosphatase buffer diluted from a 10x stock.
1x phosphatase buffer:
| A | B | |
| Tris- HCl, pH 7.4 | 50 mM | |
| NaCl | 100 mM | |
| DTT | 2 mM | |
| MnCl2 | 1 mM |
For this, protein samples were, where necessary, supplement with lysis buffer to a total volume of 45 µL and supplement with 5 µL of 10x phosphatase buffer.
The total amount of protein lysate depends on the abundance of the target protein you want to verify with western blotting. However, for us, this typically ranges between 50 µg -500 µg in total.
Incubate the samples for 00:30:00 at 30 °C before reactions were terminated by the addition of protein loading dye and heat inactivation for 00:05:00 at 95 °C .
35m
Analyse the samples by SDS-PAGE and western blotting.
Note
Note, some detergents can inhibit the phosphatase reaction, in that case opening the cells with a 26G needle can serve as a substitute in following buffer (50 mM HEPES pH 7.4, 100 mM NaCl, 2 mM DTT).