Western Blot Analysis of Immunoprecipitation (IP-Western)

IP-Western analysis remains a popular technique for identifying protein-protein interactions and identifying unknown proteins in a multi-protein complex. The steps include cell lysis, formation of the antibody-antigen (immune) complex, precipitation of the immune complexes, and analysis by Western blotting.

Click on the Western Blot Analysis of Immunoprecipitation (IP-Western) topics to read about the possible causes and remedies:

High Background



High
background,
leading to
nonspecific
bands on
Western blot
Possible Cause                Remedy

Non-specific binding to beads
  • Perform a pre-clearing step when using agarose beads. Adding non-coated agarose beads (prior to the addition of antibody-coated beads) to the protein mixture removes proteins that non-specifically bind to the agarose.
  • Ensure beads are properly blocked with BSA prior to adding antibody.
  • Decrease volume of beads.

Wash buffer not stringent enough
  • Switch to higher stringency buffer such as one that is RIPA-based.

Non-specific antibody binding
  • Decrease incubation time, lysate volume/concentration, and/or antibody concentration.
  • Use a more specific antibody.

Inadequate washing
  • Add more washes and/or wash for a longer period of time.
  • Increase stringency of the wash buffer.
  • Invert tube several times to ensure adequate washing.

No Target Protein Eluted, or Not Enough Target Protein Eluted

Possible Cause Remedy

Protein degradation, dephosphorylation, and denaturation during lysis step
  • Perform all steps at 4 °C. Ensure adequate amount of protease and phosphatase inhibitors.

Lysis buffer too stringent
  • Switch to a medium or low-stringency buffer such as one that is NP-40-, Triton® X-100-, or PBS-based.

Antibody not bound to the beads

Not enough antibody-antigen binding
  • Increase the concentration of antibody added to the lysate mixture and/or the concentration of beads.
  • Incubate antibody and lysate mixture for a longer time. 
  • In cases of low protein concentration or low antibody-protein affinity, switch from a direct immunoprecipitation method to an indirect method. 

Agarose beads accidentally removed during precipitation step
  • Ensure agarose beads are not aspirated into the pipette.
  • Use magnetic beads instead of agarose beads to minimize/eliminate bead loss.

Mouse IgG or chicken antibody does not bind efficiently to protein G- or protein-A conjugated beads (for indirect immunoprecipitation)
  • Add a bridging antibody during the precipitation step to improve the binding of these antibodies to the protein G- or protein A-conjugated beads.

Target protein remains on the beads
  • Ensure appropriate elution buffer type and pH.

Disruption of protein complexes (in co-immunoprecipitation experiments)
  • Vortex gently and use wide pipette tips to avoid disruption of protein complexes.

Extra Band(s) at 50 kDa and 20 kDa on Final Western Blot that May Mask Band of Interest

Possible Cause                          Remedy

Heavy and light chains of the primary antibody are being recognized by the secondary antibody
  • Use a secondary antibody that detects native
    (non-denatured) immunoglobulins.


In a cross-linking IP
western, the heavy
and light chains of
the primary capture
antibody are excluded
from the sample.
  • Use crosslinking IP by first crosslinking protein A or G beads to the capture antibody using DSS or similar crosslinker.

Guide to Choosing Immunoprecipitation Beads