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No protein detected during elution in immunoprecipitation (IP)

Explore potential causes and solutions for no protein detection in IP.

No eluted target protein detected

Possible causes

Solution

Insufficient antibody for capture of the target protein

  • Check that the recommended amount of antibody is being used. The concentration of antibody may require increasing for optimization of results.

Target protein has not eluted from the beads

  • Ensure you are using the correct elution buffer and that it is at the correct strength and pH for elution of the protein.

Target protein not expressed in sample used/low level of target protein expression in sample used

  • Check the expression profile of the target protein to ensure it will be expressed in the cells of your samples.

  • If there is low level of target protein expression, increase the amount of lysate used. However, this may result in increased non-specific binding so it would be advisable to pre-clear the lysate before commencing with the IP procedure.

Incorrect lysis buffer used

  • Check datasheet to see if the antibody detects denatured or native protein and ensure the correct lysis buffer is used.

Antibody has not bound to immunoadsorbant beads

  • Ensure you are using the correct beads for the antibody isotype used.