IP Troubleshooting

Troubleshooting tips and solutions for common IP problems.

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How To Improve Elution Conditions
Issue Possible solution
Wrong lysis buffer Change lysis buffer (depends on the protein of interest; nuclear or cytoplasmic protein).
No antibody binding to beads Make sure the isotypespecific beads were used.
Protein of interest cannot be eluted from the beads Change elution buffer (components, salt concentration, pH, etc).
Insufficient amount of antibody for proper binding  Optimize the antibody concentration (titration experiment).
Low expression of the protein of interest  Increase lysis volume. Pre-clean the sample to decrease non-specific binding and remove debris.


High Background Or Unwanted Protein Precipitation
Issue Possible Solution
Substances in sample bind non-specifically to either agarose/magnetic beads or antibodies Include a pre-cleaning step by incubating the lysate with Protein A/G conjugate without the antibody.
Non-specific binding to Protein A or G agarose/magnetic beads Add saturating amounts of competitor proteins, e.g., 2% BSA to agarose/magnetic beads protein-A/G.
Concentration of antibodies too high Determine the optimal concentration of antibody by titration.
Unsuited washing Use more stringent washes, e.g., 1.0 M NaCl, 0.5 M LiCl, 1 M KSCN, 0.2% SDS, or Tween 20. Consider using distilled water. Increase the number of washes. Transfer the pellet to a new tube before last washing step.
Weak/No signal
Issue Possible Solution
Antibody not capable of IP Try a different antibody. Polyclonal antibodies usually perform better than monoclonal antibodies.
Insufficient amount of primary antibody used Determine optimal concentration of primary antibody by titration experiment.
Too many competing proteins in sample Spin the lysate for 30 minutes before adding the antibody in order to remove insoluble proteins, membrane fragments, debris, etc.
Antigen of interest not present Make sure sample is suitable/appropriate for the experiment.
Antigen of interest lost or destroyed in sample Try fresh lysates. Use appropriate inhibitors for each sample preparation.
Washes were too stringent Reduce the number of washes and/or salt and detergent concentration or use a different antibody
Problems with incubation times Usually the primary antibody and antigen of interest are incubated for 4 hours to overnight at 4ºC.
Used Protein A or G may not bind species or subclass of selected primary antibody See Table 4: Affinity of Protein A and G.
Interfering substances present in sample Lysates containing dithiothreitol, 2-mercaptoethanol, or other reducing agents can destroy antibody function and should be avoided. Extremes in pH and excessive detergent concentrations may also interfere with the antibodyantigen interaction.