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
• No Target Protein Eluted, or Not Enough Target Protein Eluted
• Extra Band(s) at 50 kDa and 20 kDa on Final Western Blot that May Mask Band of Interest
• Guide to Choosing Immunoprecipitation Beads

High Background

	Possible Cause	Remedy
High background, leading to nonspecific bands on Western blot	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	Ensure beads are properly matched to the antibody isotype. Refer to guide on matching IP beads to antibody isotype
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

Guide to Choosing Immunoprecipitation Beads