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.

Read more about

• 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

Figure 1. 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	Ensure beads are properly matched to the 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

Figure 2. In a cross-linking IP western, the heavy and light chains of the primary capture antibody are excluded from the sample.

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. Use crosslinking IP by first crosslinking protein A or G beads to the capture antibody using DSS

Guide to Choosing Immunoprecipitation Beads

Relative Affinity

Antibodies	Protein A/G Mix	Protein A	Protein G	Kappa Ig Binder	Lambda Ig Binder	Kappa/Lambda Mix*
Rabbit IgG	SA	SA	SA
Mouse IgM	RE	SA
Mouse IgG3	SA	MSA	SA
Mouse IgG2b	SA	SA	SA
Mouse IgG2a	SA	SA	SA
Mouse IgG1	MSA	MSA	MSA
Human IgM	MSA	MSA		MSA	MSA	SA
Human IgE	MSA	MSA		MSA	MSA	SA
Human IgD	MSA	MSA		MSA	MSA	SA
Human IgA	MSA	MSA		MSA	MSA	SA
Human IgG4	SA	SA	SA	MSA	MSA	SA
Human IgG3	SA		SA	MSA	MSA	SA
Human IgG2	SA	SA	SA	MSA	MSA	SA
Human IgG1	SA	SA	SA	MSA	MSA	SA
Rat IgM	RE	RE
Rat IgG2c	MSA	RE	MSA
Rat IgG2b	MSA	RE	MSA
Rat IgG2a	SA	RE	SA
Rat IgG1	MSA	RE	MSA
Rat IgG	MSA	SA	SA

Fragments	Protein A/G Mix	Protein A	Protein G	Kappa Ig Binder	Lambda Ig Binder	Kappa/Lambda Mix*
Human l					SA	MSA
Human k				SA		MSA
Human Fc	MSA	MSA	MSA
Human scFv	MSA	MSA		RE	RE	RE
Human F(ab')2	MSA	MSA	MSA	MSA	MSA	SA
Human F(ab)	MSA	MSA	MSA	MSA	MSA	SA
Key code for relative affinity of protein A and G; PureProteome™ Kappa and Lambda magnetic beads for antibodies: SA = Strong Affinity; MSA = Moderate/Slight Affinity; RE = Requires Evaluation *PureProteome™ Kappa/Lambda mix is not a catalog item. Simply procure the Kappa and Lambda beads individually and mix at a 1:1 ratio.