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.
- Bead type: magnetic beads are better than any other type. They adsorb less proteins in a non-specific manner, thereby improving the Co-IP method.
- Method of purification (more accurate: enrichment): in our experience, FLAG tagging and immobilized anti-FLAG IP is the best method. If not, use an antibody that is covalently bound to beads (see below 'Crosslinking Ab to the beads'). 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).
Crosslinking an antibody to magnetic beads
- Magnetic, Protein G beads were incubated with the antibody (in PBSx1), overnight, 4deg.
- Beads were washed three times with PBSx1, and then transferred to eppendorf tubes.
- 1ml Glutaraldehyde (1%) in PBSx1 (DO NOT USE: Tris or any amine containing solution, as it will quench the glutaraldehyde) was added for 15 minutes, incubated at RT, vigorous shaking. Large volume (1ml) is needed to keep the beads apart.
- Add TBSx1 to fill the tube.
- Wash beads twice with TBSx1, incubate for 5 minutes to quench the reaction.
- Add the lysate (TBSx1, Triton 1%, Tween 0.1%), at RT for ~4hrs; at 4deg for ON.
- Wash x3-5 in the same lysis buffer, large volume (1ml), chilled.
- Then 25-100µl (50mM Tris pH 7.5 - SDS 5%) buffer at 25°C with gentle shaking for 10 minutes. (Do not add reducing agent).
- Transfer the supernatant to new eppendorf tube, freeze in -80deg and send to the Protein Profiling unit, frozen.