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Immunoprecipitation Assay to Study Enzyme Receptor Interactions in Transfected Cells
In This Article
Overview
In this video, we demonstrate a technique to perform the co-immunoprecipitation of GFP-tagged wild-type and mutated PD-1 proteins using beads coated with anti-GFP antibodies. The bead-bound PD-1-GFP proteins are incubated with SHP2 proteins that interact with PD-1, enabling the identification of specific structural motifs in PD-1 that play a critical role in SHP2 interaction.
Protocol
1. Immunoprecipitation
NOTE: The following steps should be performed on ice or at 4 °C.
- Supplement the lysis buffer (50 mM Tris-HCl at pH 7.2, 250 mM NaCl, 0.1% NP-40, 2 mM EDTA, 10% glycerol) with protease inhibitors (dissolve 1 tablet in 10 mL of lysis buffer) and with 1 mM sodium orthovanadate. Add 500 µL of ice-cold lysis buffer to the cells and remove and collect the cells from the plates immediately using a cell scraper.
NOTE: It is important to add the sodium orthovanadate only to the lysis buffer that will be used for the PD-1-GFP-transfected plates since the SHP2-transf....
Representative Results
Figure 1: Experimental conditions and strategy. GFP-PD-1 WT (wild type), GFP-PD-1 Y223F (ITIM mutant), or GFP-PD-1 Y248F (ITSM mutant) were expressed in HEK 293T cells that were subsequently treated with pervanadate. Phosphorylated GFP-PD-1 proteins collected by GFP immunoprecipitation were mixed with lysates from cells overexpressing SHP2, and the levels and activity of SHP2 bound to each vers.......
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Source: Strazza, M., et al. Co-immunoprecipitation Assay for Studying Functional Interactions Between Receptors and Enzymes. J. Vis. Exp. (2018).
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