Immunoprecipitation Assay to Study Enzyme Receptor Interactions in Transfected Cells

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.

1. 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-transfected plates and non-transfected control plates must retain phosphatase activity.
2. Transfer the lysates into 1.5 mL cold tubes and rotate them at 0.005 x g, 4 °C for 30 min.
3. To collect the post nuclei supernatant (PNS) from the lysates, spin down the lysates for 10 min at 10,000 x g at 4 °C and transfer the supernatants into new tubes. Discard the pellet. Store the supernatants of the second set of six plates (Figure 1) on ice for later whole-cell lysate analysis.
4. Preparation of the anti-GFP beads for immunoprecipitation of PD-1-GFP from the PD-1-GFP-transfected lysates of the first set (four plates) (Figure 1).
   1. To prevent the settling of the beads, gently shake the bottle with the beads before opening. Remove 40 µL of the anti-GFP beads from the slurry per each condition.
   2. Spin at 500 x g for 3 min at 4 °C and remove the supernatant to wash the beads.
      NOTE: It is important to minimize the contact between the pipette plastic tips and agarose beads to prevent loss. It is recommended to cut the edge of a 200 µL tip before transferring the beads to another tube.
   3. Resuspend the beads in 80 µL of lysis buffer (per sample).
5. Add the washed beads directly to the cell lysate (PNS) from the PD-1-GFP-expressing cells of the first set (four plates) (step 1.3). Rotate at 0.005 x g for 30 min at 4 °C to immunoprecipitate the GFP-tagged proteins.
6. Wash the beads with 1 mL of cold lysis buffer (without orthovanadate) 3 times. Centrifuge the tube at 2,500 x g for 10 s.
   NOTE: When adding the lysis buffer to the beads, add it directly onto the beads without touching them with the tips. There is no need to pipette up and down during the washes.
7. Equally, divide the lysate of the active SHP2 from the first set (one plate; step 1.3) and add it to three tubes of the washed PD-1-GFP-containing beads (the WT PD-1-GFP and the two different phospho-deficient mutations, Y223F and Y248F). Add one-third of the volume from the lysate of the non-transfected cells to the second tube of the WT PD-1-GFP beads. Discard the remaining two-thirds.
8. Incubate the beads for 4 h at 4 °C with gentle rotation (0.005 x g).
9. Wash the beads twice with 1 mL of cold lysis buffer (without pervanadate or orthovanadate), as reported in step 1.6.
10. Add 80 µL of lysis buffer (without pervanadate or orthovanadate) per sample ensuring that the total volume in each tube is 100 µL (20 µL of beads and 80 µL of lysis buffer). Mix gently and transfer 50 µL from each tube into two fresh 1.5 mL tubes.
    NOTE: Following this step, there will be two tubes filled with a mixture that contains beads and lysis buffer. One of the tubes will be used for testing the co-immunoprecipitated SHP2 by western blotting.

2. SHP2 Western Blot Analysis

1. Spin down the beads and remove the supernatant.
2. Add 20 µL of 2x Laemmli buffer (see Materials Table) to the beads and boil at 95 °C for 5 min.
3. Measure the protein concentration of the input controls (second set) using a BCA kit (see Materials Table). Transfer 30 µL of the most diluted sample to a new tube. Dilute the rest of the input controls with lysis buffer to the same concentration as the most diluted sample and transfer 30 µL from each of them to a new tube.
   NOTE: Following this step, there will be 4 tubes with 30 µL each, at similar protein concentrations, representing the input controls lysates.
4. Add equal volumes of 2x Laemmli buffer to the lysates and boil at 95 °C for 5 min. Spin down the beads and load the supernatant on 4–20% SDS-PAGE Tris-based gel (see Materials Table) for western blot analysis. In addition, load 50 µL from each sample of the second set.
5. Continue with western blot analysis.

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 version of PD-1 were recorded.

Materials

Name	Company	Catalog Number	Comments
Nitrocellulose membranes	General Electric	10600004
Microcentrifuge	Eppendorf	5424-R	1.5 mL
BCA	Fisher	23225	Bi Cinchoninic Acid assay
Anti-SHP2	Santa Cruz	SC-280
Anti–GFP-agarose	MBL	D153-8
Anti-GFP	Roche	118144600
Anti-Actin	Santa Cruz	SC-1616
HEK-293 cells	ATCC	CRL-1573
Orthovanadate	Sigma	S6508
Protease inhibitor cocktail	Roche	11836170001	EDTA-free
Tris-Glycine SDS Sample Buffer (2x)	Invitrogen	LC2676	Modified Laemmli buffer
4-20% Tris-Glycine Mini Gels	Invitrogen	XP04205BOX	15-well
Trypsin-EDTA (0.25%) Phenol Red	Gibco	25200114
NaCl	Sigma	S7653	Sodium chloride