3. Antibody Fragments Blocking the Signal of Interest

In some cases, detection of the antibody used to pull down sample components during the IP or co-IP will interfere with interpretation of the results from a Western blot or during mass spectrometry analysis. There are two major methods to avoid this problem: antibody immobilization or a detection reagent that does not recognize the denatured antibody fragment.

1. Antibody immobilized on magnetic beads

Protein A, G or L binding of antibody followed by covalent attachment with a cross linker; orients the antigen binding sites outward, but can result in significant loss of antibody function. This choice is compatible with unpurified antibodies. Directly immobilize antibody onto an activated magnetic beads can greater preserve the antibody's function. This choice requires a purified antibody because the resin reacts with amines found on all proteins.

2. Detection reagent specific to native antibody

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1. No Proteins Detected following IP
2. High Background (Specific or Nonspecific)
3. Antibody Fragments Blocking the Signal of Interest
4. The Worst Case Scenario: Co-IP is Not Successful
Immunoprecipitation / IP FAQ