How do you use a co-IP?

The general steps are as follows:

1. Lyse your Cells. In this step you gently break open your cells to make your protein accessible to the antibody.
2. Add your Antibody.
3. Add the Protein A/G Beads.
4. Incubate.
5. Collect.
6. Wash the Beads.
7. Elute your Protein(s)
8. Detect your Protein(s)

What is IgG control for IP?

Normal Rabbit Control IgG is essential for ELISA, Western Blot (WB), Immunohistochemistry (IHC) and Immunoprecipitation (IP) experiments. It’s purpose is to estimate that the proteins stained in the experiment result are due to the specific interaction with the antibody.

How do I preclear lysate IP?

The basic approach to preclear a lysate is to incubate the sample with exactly the same components that will be used for the immunoprecipitation, except use a nonspecific antibody from the same host species as the IP antibody. Any nonspecific immune complexes will form and be immobilized to the beaded support.

What does co-IP tell you?

Co-immunoprecipitation (co-IP) is a popular technique to identify physiologically relevant protein–protein interactions by using target protein-specific antibodies to indirectly capture proteins that are bound to a specific target protein.

When to use preclearing beads for immunoprecipitation?

Preclearing is designed to remove potentially reactive, non-specific components from a lysate sample prior to the actual immunoprecipitation procedure. Preclearing is frequently necessary when using agarose beads, but it is seldom required when using magnetic beads (see videos below).

What do you need to know about Co-IP elution?

Elution:Usually accomplished with very harsh conditions such as boiling of the beads in a Reducing SDS- Sample Loading Buffer. When using immobilized antibodies, milder conditions (pH-shift) are applied to avoid disruption of the antibody. In this case, only antigen (antigen and binding partners for co-IP) are eluted free of antibody contamination.

What kind of blotting is needed for Co-IP?

In this case, only antigen (antigen and binding partners for co-IP) are eluted free of antibody contamination. Detection: Typically performed by Western blotting. In co-IP experiments often very small amounts of protein are available for detection, so high-sensitivity Western blotting substrates are required.

How to check positive control in Co-IP?

To allow optimization, always check the following control samples together with the desired eluate: • Lysate in IP-Buffer — positive control confirms sample contains protein of interest. If not detectable, your sample may not have the protein of interest.