Phosphoflow Protocol

A commonly used quantifiable metric for protein phosphorylation is achieved through the use of fluorescence tagged antibodies specific for phosphorylated protein sites. Post treatment cell samples incubated with these antibodies can be analyzed via flow cytometry to measure their respective levels of phosphorylation. Be sure to include unstained controls and secondary antibody only controls if necessary.

  1. Treat 0.5-2x106 cells per condition for 2-3 hours.

  2. Collect cells to conical tubes / eppitubes and spin down cells (500g, 5 minutes).

  3. Prechill 810µl Methanol in -20 C freezer.

  4. Resuspend pellet in 100µl PBS (does not have to be sterile).

  5. Add 100µl of 4% paraformaldehyde (PFA) -or- 25µl of 16% PFA to each sample.

  6. Incubate 10 min at room temperature.

  7. Add 800µl of ice cold methanol 100% methanol. At this stage, you can store at -20oC indefinitely or incubate samples for 30 minutes before proceeding to the next step.

  8. Spin down cells 12,000rpm, 5min. This spin should be as fast as possible to ensure maximum sample collection.

  9. Add 1 ml PBS to each sample.

  10. Spin down cells 12,000rpm, 5min.

  11. Prepare 100 uL of primary antibody master mix per sample. Concentrations may need to be 2-5 times higher than an equivalent western blot procedure.

  12. Discard sample supernatant and add 100µl of diluted antibody in PBS.

  13. Incubate 30 minutes in the dark on ice (or a fridge).

  14. Wash by adding 1 mL PBS and incubate for 5 minutes at room temperature. Spin down cells at 8000 rpm, 5 minutes. Prepare secondary antibody solution in the meantime if necessary (100 uL solution per sample).

  15. Remove supernatant. If using secondary antibody repeat resuspension, incubation, and wash steps with secondary antibody solution. If proceeding to FACS analysis, resuspend in 250 uL PBS.

  16. Analyze via FACS.

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