In vitro phosphatase assay

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 300x g,4°C.

After a PBS wash to remove the remaining cell medium, resuspend the cell pellets in lysis buffer.

Lyse the samples for 0h 20m 0s On ice. Clear the cell lysates by centrifugation at 20000x g,4°C.

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 100µg of cell lysate with 5µL of 10x NEBuffer for Protein MetalloPhosphatases (P0753, New England Biolabs) and 5µL of 10millimolar (mM) of MnCl₂ to make a total reaction volume of 50µL.

Add 1µL 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 0h 5m 0s.

Analyze the samples by western blot analysis as described above.