IP Troubleshooting

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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.