Immunoprecipitation FAQ

Frequently asked questions for IP

  1. How much antibody and how much sample should be used?
  2. When running the IP purified protein on a western blot, there are two extra bands. Is this the antibody eluting with the protein? How can this be avoided?
  3. I would like to do immunoprecipitation using an IgM antibody. How do I do this as IgM does not bind protein A or G?

1. How much antibody and how much sample should be used?

We recommend to use 10-500 μg cell lysate per sample plus the recommended amount of antibody. These amounts will be chosen depending on the abundance of the protein and the affinity of the antibody for the protein, typically in a pilot experiment where a fixed amount of protein is precipitated by increasing amounts of antibody.
You can check the antibody datasheet for recommended antibody concentration. As a guideline use:

1 – 5 μl polyclonal antiserum
1 μg affinity-purified polyclonal antibody
0.2 to 1 μl ascites fluid (monoclonal antibody)
20 to 100 μl culture supernatant (monoclonal antibody)

2. When running the IP purified protein on a western blot, there are two extra bands. Is this the antibody eluting with the protein? How can this be avoided?

To enable elution of protein with little antibody contamination (for cleaner protein preparation and cleaner western blots), it is recommended to cross link the antibody to the beads. The target protein should then be eluted with a mild eluent, such as glycine buffer.  There is a guideline procedure available:

Crosslinking antibody to the beads protocol

3. I would like to do immunoprecipitation using an IgM antibody. How do I do this as IgM does not bind protein A or G?

You can use protein L for IgM immunoprecipitation. Or you can attach the IgM antibody to the protein A or G beads using an IgG anti-IgM antibody. See our guidelines for IP using IgM antibodies

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