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How do you lyse cells for co-IP?

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How do you lyse cells for co-IP?

For Co-IP of soluble proteins, use a non-detergent, low-salt lysis buffer. This mild lysis buffer is probably least likely to interfere with protein-protein interactions. For less soluble protein complexes, add non-ionic detergents such as Nonidet™ P40 Substitute or Triton™ X-100 to lysis buffer.

How do you normalize co immunoprecipitation?

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  1. Use equal amounts of starting protein.
  2. Use equal amounts of IP antibody.
  3. Use equal amounts of capture beads/resin.
  4. Elute with equal volumes.
  5. Load equal volumes.
  6. Blot as usual for the bait (IP protein) and prey (interacting proteins)
  7. Quantify the both the bait and prey protein signal.

Can I add SDS when doing co-IP?

In theory you should add SDS buffer at the same time to both your IP and input to account for degradation products/ other time dependent things but in practice you can add it to your input whenever as long as the ug stays the same for your experiment.

How do you increase immunoprecipitation efficiency?

The smaller the volume, the more effective your IP works. Using a small volume keeps your protein concentration high and therefore increases the binding affinity. Concentration is a function of volume. Try to use a volume as small as possible.

How much protein do you need for co IP?

So basically cell lysate protein content of 10µg/µL is OK. However, majority of proteins do not have that high expression in cells. Therefore, 500-1000 microgram will be good starting point if you do not know the expression level of your protein of interest.

How much protein should I load for immunoprecipitation?

Use 25 µl of Protein A or Protein G Magnetic Beads per 200 µl of crude cell lysate containing 200-500 µg of total protein in a standard immunoprecipitation protocol. It is important to increase the volume of beads proportionately for larger cell lysate volumes.

How do you make a Coip?

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)

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