Incubation of antibody-agarose beads:
When cells are lysed and pellet is discarded after centrifuge, the supernatant can be stored at -80°C for 3 months. It’s better to use freshly prepared supernatant of cell lysate to be applied in incubation of antibody-agarose beads.
Antibodies can be added in the supernatant and incubate for hours, then added protein A or G with beads for an overnight incubation. Alternatively both antibodies and protein A/G-beads are added in the supernatant.
Generally, 1μg antibody for 1mg/ml protein, and not exceed 5μg. Over dose antibody will lead to false positive results.
In this step the key point is choosing a suitable negative control. Normally adding in the same amount of IgG is as the negative control. A more convincing method is that one chooses a non-relative protein’s antibody. For example, if your target protein is membrane protein A, you can choose a membrane protein B as a negative control (add in protein B’s antibody) as long as you make sure protein A and protein B won’t interact. As the same, if your target protein is a cytoplasmic soluble protein C, choose another cytoplasmic soluble protein D.
To avoid another false positive result possibility caused by non-specific binding of protein A/G-beads, before adding in antibody of target protein, you can pre-incubate protein A/G-beads with cell lysate for couple of hours, then incubate the supernatant with antibody-agarose beads.
Protein A and G have different affinities for different antibodies (e.g. species and Ig subtype of primary antibodies). Therefore adding in both protein A and G can get this problem solved.