Immunoprecipitation Assay to Study Enzyme Receptor Interactions in Transfected Cells

Programmed death-1, PD-1, a cell surface receptor protein, contains two cytoplasmic tyrosine motifs — the inhibitory motif, ITIM, and the switch motif, ITSM. Ligand binding induces ITIM and ITSM phosphorylation and tyrosine phosphatase protein SHP2 recruitment to the cytoplasmic domain of PD-1, triggering a downstream signaling cascade.

To study SHP2-PD-1 interactions via a co-immunoprecipitation assay, obtain an adherent epithelial cell culture. These cells express green fluorescent protein, GFP-tagged wild-type PD-1 with phosphorylated ITIM and ITSM. Obtain epithelial cells expressing GFP-tagged mutant PD-1, wherein ITIM or ITSM mutations inhibit their phosphorylation.

Add lysis buffer containing detergent and sodium orthovanadate. The detergents lyse the cells, causing GFP-tagged PD-1 protein release. Sodium orthovanadate blocks released phosphatases, thus, preventing ITIM and ITSM dephosphorylation.

Centrifuge the lysate. Transfer the protein-containing supernatant into fresh tubes. Add anti-GFP antibody-coated agarose beads. The GFP-tagged PD-1 proteins bind to the anti-GFP antibodies.

Incubate with the SHP2 suspension. The bead-bound PD-1-GFP binds SHP2 via the phosphorylated