Co-immunoprecipitation of synaptic proteins

Proteins at a synapse rarely work alone: receptors are held in place by scaffold proteins, which in turn bind enzymes and other partners. PSD-95 is a prominent protein of the postsynaptic density, the protein-dense layer under the membrane of excitatory synapses (Cho et al., 1992). Binding studies with fragments of the two proteins in yeast and in the test tube showed that the end of the intracellular tail of the NMDA receptor subunits GluN2A and GluN2B (in older papers NR2A and NR2B) binds the PDZ domains of PSD-95 (Kornau et al., 1995; Niethammer et al., 1996).

Co-immunoprecipitation (co-IP) asks whether two proteins are associated in a tissue. The tissue is dissolved in a detergent buffer, an antibody against one protein (the bait) is added, and beads coated with protein A or protein G, bacterial proteins that bind antibodies (Forsgren & Sjöquist, 1966; Björck & Kronvall, 1984), pull the antibody out of solution with whatever is attached to it. A Western blot then shows whether the other protein (the prey) came with it. PSD-95 has been co-immunoprecipitated with GluN2A and GluN2B from rat hippocampus dissolved in deoxycholate (Takagi et al., 2000), and GluN2B also associates with other members of the PSD-95 family: in adult rat hippocampus it appears to be found preferentially with SAP102 (Sans et al., 2000).

The method is simple to describe and easy to get wrong. The postsynaptic density resists mild detergents (Carlin et al., 1980), while strong detergents break complexes apart; proteases cut GluN2B (Simpkins et al., 2003); the antibody itself appears on the blot; and molecules can meet in the tube after lysis, as shown for an RNA-binding protein and its target RNA (Mili & Steitz, 2004). A co-IP also shows only that two proteins were in the same complex: PSD-95 complexes from brain contain dozens of proteins (Husi et al., 2000; Husi & Grant, 2001).

Unlike in a real laboratory, the true state of the tissue is known in the simulation, and the final station shows which decisions changed the result.

Learning objectives

• Design a co-IP with the animal or donor as the unit, each lysate split between the specific IP and its controls
• Choose a lysis buffer by weighing solubilisation against the survival of complexes, and protect proteins with inhibitors and cold
• Choose an IP antibody, protein A or G beads and the controls a co-IP needs: input, isotype IgG, beads only and, where possible, knockout tissue
• Read a co-IP blot, including antibody heavy and light chains at approximately 50 and 25 kDa, and confirm the result with a reciprocal IP
• State what a co-IP can and cannot show: association in a complex, not direct binding, and the possibility of association after lysis
• Quantify percent of input and compare the IP with its control by a paired test across animals or donors

Ethics and safety

Animal tissue comes from mice or rats used under a protocol approved by an Institutional Animal Ethics Committee (in India, the CCSEA under the Prevention of Cruelty to Animals Act 1960; elsewhere, frameworks such as EU Directive 2010/63/EU, the US Animal Welfare Act and PHS Policy, and the UK Animals (Scientific Procedures) Act 1986); knockout controls add animals, so group sizes are justified in advance and tissue is shared between experiments where possible (Reduction, one of the 3Rs), and experiments are reported following the ARRIVE guidelines, developed by the NC3Rs in the United Kingdom and endorsed by journals internationally (Percie du Sert et al., 2020). Human tissue comes from brain banks, from donors who gave consent, and its use needs approval from an ethics committee (in India, an Institutional Ethics Committee following the ICMR National Ethical Guidelines of 2017; elsewhere, a research ethics committee or, in the United States, an institutional review board); nothing can be manipulated, and groups can only be defined by diagnosis or circumstances. Its quality depends on the agonal state, the post-mortem interval and storage (Ferrer et al., 2008). A co-IP needs fresh-frozen, unfixed tissue, because fixation cross-links proteins. At the bench, detergents, SDS, reducing agents and the electrophoresis equipment require laboratory safety training, gloves and eye protection; human tissue is handled as potentially infectious under the institution's biosafety rules.

Key terms

• Co-immunoprecipitation (co-IP): Pulling one protein (the bait) out of a tissue extract with an antibody and testing whether another protein (the prey) comes with it.
• Lysate: The liquid obtained when tissue is broken up in a detergent buffer and the insoluble debris is spun down; it contains the dissolved proteins.
• Detergent: A molecule that dissolves membranes. Ionic detergents (SDS, deoxycholate) are strong and can pull complexes apart; nonionic ones (NP-40, Triton X-100) are mild.
• Postsynaptic density: The dense layer of proteins under the membrane of an excitatory synapse, including PSD-95 and glutamate receptors; it resists mild detergents.
• PDZ domain: A protein module of about 90 amino acids that binds a short motif at the very end of another protein; PSD-95 has three.
• Protease and phosphatase inhibitors: Chemicals that block enzymes which cut proteins or remove their phosphate groups once the cells are broken.
• Pre-clearing: Incubating the lysate with beads alone and discarding them, to remove proteins that stick to beads before the IP.
• Isotype control: An unrelated antibody of the same species and class as the IP antibody, used at the same amount, to measure non-specific binding.
• Input: A small, known fraction of the lysate loaded directly on the gel, showing what was present before the IP.
• Protein A and protein G: Bacterial proteins that bind the constant part of antibodies; coupled to beads, they collect the antibody and what it holds.
• Epitope: The part of a protein that an antibody binds. If it overlaps the binding site of a partner, the antibody may capture only the free protein.
• Heavy and light chains: The two kinds of chain of an IgG antibody, approximately 50 and 25 kDa after reduction; eluted IP antibody shows as bands at these sizes.
• Reciprocal IP: The same experiment with the roles swapped: the former prey becomes the bait.
• Percent of input: The prey in the IP lane as a percentage of the prey in the lysate used for one IP, calculated from the input lane.