Immunoprecipitation troubleshooting

Problems:
No binding
High background signal


No binding

• Target protein not present in the cell extract, lost or distroyed during first step of preparation.
  Check that protein of interest was actually present in the cell extract/lysate. Prepare fresh extracts, do not freeze them. Use suitable protease inhibitors.
  
  
• Washes are too strong.
  Decrease the amount of washing step as well as cncentration of detergent and salt.
  
  
• Large number of competing proteins present in the sample.
  Centrifugate cell extract/lysate at 10 000 x g for 30 minutes before adding antibody. This should help to remove unsoluble aggregates.
  
  
• Interfeering substances present in the cell extract/lysate.
  Make sure that no DTT, mecraptoethanol or other reducing agents are added to your lysates. They will be a reason for antibody breakdown. Extreme pH and increased detergent concentrations might also interfere with establishing of the antigen-antibody complex.
  
  
• Wrong beads used to isolate the antigen-antibody complex.
  Check which beads Protein A, G, will bind best class of antibodies you are working with.
  
  
• Antibody not able to perform immunoprecipitation or used in too low amount.
  Precipitating antibody has to be changed to another one. Usually polyclonal antibodies are better in immunoprecipitation assays than monoclonal antibodies. Amount of the antibody used in this assay has to be titrated. In some cases, if for instance antibodies were rised against denatured antigen, native epitopes might not be recognized.
  
  
• Is your target protein glycosylated? Glycosylation can contribute to steric hindrance of the epitope and unable the antibody to reach it, by simply masking it. You can try to denature the protein by heating up the whole extract for several minutes. Although, it will also contribute to agregate formation of other proteins present in the extract.

• Antibody concentration used is too high.
  Titrate antibody used.
  
  
• Non-specific binding of other proteins either to the Protein A or G beads or to the antibodies.
  
  
  

• SeizeTM X Immunoprecipitation Kits (Pierce)
  
• Dynal beads (Dynal)
  
  Both will allow to couple pernamently primary antibody and bypass the problem with light or heavy immunoglobulin chains which might interfere with the detection of a target protein following gel electrophoresis.