Co immunoprecipitation and pulldown experiments (also known as affinity purification mass spectrometry AP-MS) are typically conducted to identify protein-protein interactions or protein DNA/RNA interactions. There are many ways to conduct such experiments and many things to consider. We tried to summarize the key factors for a successful experiment:
- Controls: Co-IP and pulldown experiments include many non-specific interactions. The ONLY way to know whether a hit is a potential interactor is by reference to a negative control.
- Method of purification (more accurate: enrichment): in our experience, FLAG tagging and immobilized anti-FLAG IP is the best method. If not, use immobilized antibody against your bait. Incubation of the antibody followed by protein A/G purification is much less affective.
- Lysis: one must consider two things when choosing the lysis buffer.
3.1.An efficient extraction of the proteins.
3.2. Not to breakdown the interactions between the proteins by using strong or high concentration of detergents or denaturants.
- Protein loading: the amount of protein you load depends on:
4.1 The binding efficiency of the antibody.
4.2 The abundance of the bait protein.
4.3 The amount of beads (antibody) you use.
4.4 The best way is to calibrate the loading and antibody amounts, though usually this is not practical to do.
4.5 Generally, protein loading ranges from 0.5mg to 10mg and bead slurry from 10 to 300uL.
- Elution: Our preference is to elute with 5% SDS (in 50 mM TrisHCl, pH 7.4). We then use the S-trap method for digestion of the eluted proteins. This is true for most tags, including FLAG and HA. We can also perfrom on-bead digestion for pulldown experiments, such as biotin-strepavidin.
- Washes: for on-bead digestion, a prerequisite is that the last two washes (after loading the lysate onto the beads) are without detergents (PBS is fine).