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 ITIM and ITSM.

Add Laemmli buffer and boil so that the reducing agents and detergents in the buffer denature the PD-1-GFP-SHP2 protein complexes. Centrifuge and collect the supernatant containing the denatured protein complexes. Perform SDS-PAGE and western blot.

Wild-type and ITIM-mutated PD-1 interacts with SHP2. ITSM mutants fail to bind SHP2, indicating that the PD1-SHP2 interaction requires phosphorylated ITSM.

While performing immunoprecipitation, all steps should be performed either on ice or at 4 degrees Celsius. Supplement the lysis buffer with protease inhibitors by dissolving one tablet of the inhibitors in 10 milliliters of buffer. Also, add 1 millimolar of sodium orthovanadate to the buffer that will be used on the PD1-GFP transfected plates. Add 500 microliters of ice-cold lysis buffer to the cells, making sure to add the lysis buffer containing the sodium orthovanadate to only the plates containing PD1-GFP transfected cells.

Use a cell scraper to immediately remove and collect the cells from the plates. Transfer the lysates into 1.5-milliliter cold tubes, and rotate them at 0.005 times g and at 4 degrees Celsius for 30 minutes. To collect the post-nuclei supernatant from the lysates, spin them down at 10,000 times g and 4 degrees Celsius for 10 minutes.

Transfer the supernatants into new tubes and discard the pellet. Store the supernatants for the second set of six plates on ice, for later WCL analysis.

To begin preparing anti-GFP beads, gently shake the bottle containing the beads before opening to prevent settling. Remove 40 microliters of the anti-GFP beads from the slurry per each condition. Centrifuge at 500 times g and 4 degrees Celsius for 3 minutes. Remove the supernatant to wash the beads, making sure to minimize contact between the pipette and the beads to prevent loss.

Resuspend the beads in 80 microliters of lysis buffer per sample. Add the washed beads directly to the cell lysate from the PD1-GFP-expressing cells of the first set of four plates. Rotate at 0.005 times g for 30 minutes at 4 degrees Celsius to immunoprecipitate the GFP-tagged proteins. Wash the beads three times using 1 milliliter of cold lysis buffer that does not contain orthovanadate for each wash. Then, centrifuge at 2,500 times g for 10 seconds.

Divide the lysate of the active SHP2 from the first set of plates into three equal portions, and add a portion to each of the three tubes containing the washed PD1-GFP-containing beads. Add 1/3 of the lysate volume from the non-transfected cells to the second tube of the wild-type PD1-GFP beads. Discard the remaining 2/3rds.

Incubate the beads for 4 hours at 4 degrees Celsius with gentle rotation at 0.005 times g. After this, wash the beads twice, using 1 milliliter of cold lysis buffer for each wash. Add 80 microliters of lysis buffer to each sample, ensuring that the total volume in each tube is 100 microliters. Using a cut pipette tip, pipette gently up and down to mix. Then, transfer 50 microliters from each tube into two fresh 1.5-milliliter tubes.

First, spin down the beads at 2,000 times g and 4 degrees Celsius for 30 seconds and remove the supernatant. Add 20 microliters of 2x Laemmli buffer, and boil at 95 degrees Celsius for five minutes. Using a BCA kit, measure the protein concentration of the input controls.

Transfer 30 microliters of the most diluted sample to a new tube. Use lysis buffer to dilute the rest of the input controls to the same concentration as the most diluted sample. Then, transfer 30 microliters of each of the diluted input controls to a new tube. Add equal volumes of 2x Laemmli buffer to the lysates, and boil them at 95 degrees Celsius for five minutes.

After this, spin down the beads at 2,000 times g and 4 degrees Celsius for 30 seconds before performing Western blot analysis as outlined in the text protocol.