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Immunoprecipitation (IP) Troubleshooting Guide

  • How to fix no binding in IP?

    Low immunoprecipitation (IP) efficiency is commonly caused by insufficient antibody, overly stringent washing conditions, incorrect Protein A/G/L bead selection, or poor target protein expression in the sample. Optimizing antibody conditions, lysis buffer composition, and wash stringency can help improve target protein pull-down and IP performance.

     

    Common causes of no binding in IP assay and troubleshooting solutions
      Possible Causes What You Can Do
    Insufficient antibody
    Check the recommended amount of antibody as indicated in the datasheet.  Titrate and optimize the optimal antibody amount used per IP experiment.
    Washes too stringent
    		 Reduce the number of washes. Reduce salt concentration in the wash buffer.
    Incorrect Protein A/G/L used
    Make sure that the Protein A/G/L beads are capable of binding to the antibody subclass being used.
    Target protein not present in the
    sample used    		 Make sure that the target protein is expressed at a relatively high level in the sample used by including an Input sample in the WB.
    Incorrect Lysis buffer used
    Make sure that the lysis buffer used is not over-denaturing and destroy the native conformation of the target proteins.
    Antibody not capable of
    immunoprecipitation
    		 Try a different antibody. Try polyclonal antibody if monoclonal antibody does not work well.

     

  • How to reduce heavy and light chain interference in IP?

    Heavy and light chain interference in IP is most commonly caused by the secondary antibody cross-reacting with denatured IgG from the primary antibody. To resolve this, use a secondary antibody that recognizes only the native form of IgG for immunoblotting.

     

    Cause and solution for heavy and light chain interference in IP western blot
      Possible Cause What You Can Do
    Secondary antibody recognizes denatured heavy and light chains from the primary antibody Use a secondary antibody that recognizes only the native form of IgG for immunoblotting.

  • How to Troubleshoot High Background in IP?

    High background in immunoprecipitation (IP) is most commonly caused by inadequate washing, excessive antibody concentration, non-specific binding to Protein A/G/L or agarose beads, or sample degradation.

    To reduce background, optimize washing stringency and antibody concentration. Non-specific binding may be reduced by bead pre-blocking or a pre-clear step. For degraded samples, protease and phosphatase inhibitors should be included during sample preparation.

     

    Common causes of high background in IP assay and how to reduce nonspecific binding
      Possible Causes What You Can Do
    Inadequate washing
    Use a more stringent washing buffer. Try to  use a high salt washing buffer or add 0.2% SDS or 1% Tween20 to washing buffer. Increase the number of washes.
    Concentration of antibodies too high Check the recommended amount of antibody as indicated in the datasheet.  Titrate and optimize the optimal antibody amount used per IP experiment.
    Non specific binding to Protein A,G or L Pre-block beads with BSA. Incubate beads with 2%BSA in PBS for 1 hour wash in PBS before use.
    Non specific binding to agarose beads Include a pre-clear step by incubating lysate with Protein A/G/L agarose beads.
    Antibody not specific enough Use affinity purified and pre-absored antibody for IP experiments.
    Sample degradation Add adequate protease inhibitors and phosphatase inhibitors throughout sample preparation and Ip steps.