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. Author manuscript; available in PMC: 2016 May 10.
Published in final edited form as: Methods. 2014 Nov 15;77-78:164–171. doi: 10.1016/j.ymeth.2014.11.008

Fig. 5.

Fig. 5

Immunoprecipitation PTEN phosphatase assays (18) MEFs are washed in PBS, lysed, and centrifuged at 16,000g for 30 min at 4 °C. Lysates are pre-cleared with 2 μg rabbit IgG plus 40 μL protein A/G beads for 1 h at 4 °C. PTEN is immunoprecipitated with 138G6 antibody plus 40 μL protein A/G beads overnight at 4 °C. Beads are washed with phosphatase buffer and incubated with 20 μM soluble di-C8-D-myo-PIP3 for 30 min at 37 °C. The beads are removed from the reaction mixture by low speed centrifugation, and the supernatant is added to 100 μL Biomol Green reagent for 15 min. The absorbance level at 620 nm is the measure of free phosphate released by PTEN.