Troubleshooting of Immunoprecipitation (IP)

Here Creative Biolabs provides the common immunoprecipitation (IP) troubleshooting tips. Though the tips provided here cover many different problems you may encounter in IP assays, we hope that you will find the information beneficial to you and useful as a reference guide. If you ever need more assistance with your experiments, please contact us for additional help. Protocol of Immunoprecipitation (IP) is available for you.

Contents

High Background
Weak or No Signal
High Amount of Antibody Eluting
No Target Protein Eluted, or Not Enough Target Protein Eluted
Co-IP Not Successful

High Background

Possible Cause	Recommended Solution
Incomplete washing	Wash well at relevant stages by placing a lid on the tube and inverting several times before centrifuging.
Non-specific proteins are binding to the beads	Beads are not pre-blocked enough with BSA. Incubate fresh beads for 1 h with 1% BSA in PBS. Wash 3-4 times in PBS before using them.
Carry over of proteins that are not detergent soluble	Remove supernatant immediately after centrifugations.
Antibody used contains antibodies that are not specific enough	Use an affinity purified antibody.
Too much antibody used leading to non-specific binding	Use less antibody.
Too many cells/too much protein in lysate leading to a lot of non-specific proteins in eluate	Reduce the number of cells/lysate used.
Non-specific binding of proteins to antibody	Reduce the amount of sample loaded onto the beads. Pre-clear the lysate by pre-incubating the prepared lysate with the beads before commencing with the IP.
Antigen degrading during IP	Ensure fresh protease inhibitors are added.

Weak or No Signal

Possible Cause	Recommended Solution
Antibody not capable of IP	Polyclonal antibodies (pAbs) usually perform better than monoclonal antibodies (mAbs).
Insufficient primary antibody	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 to remove debris, insoluble proteins, membrane fragments, etc.
Antigen of interest lost or destroyed in sample	Try fresh lysates. Use appropriate inhibitors for each sample preparation.
Used protein A or G may not bind species or subclass of selected primary antibody	Choose correct beads.
Washes too stringent	Reduce the number of washes and/or salt and detergent concentration.

High Amount of Antibody Eluting

Possible Cause	Recommended Solution
Too much antibody eluting with the target protein	Crosslinking the antibody to the beads before the IP. Eluting using a gentle glycine buffer gradient should significantly reduce the amount of antibody eluted.

No Target Protein Eluted, or Not Enough Target Protein Eluted

Possible Cause	Recommended Solution
Target protein not expressed in sample used/Low level of target protein expression in sample used	Check the expression profile of the target protein. For low level of target protein expression, increase the amount of lysate used and pre-clear the lysate before commencing with the IP procedure.
Insufficient antibody for capture of the target protein	Check that the recommended amount of antibody is being used.
Target protein has not eluted from the beads	Ensure you are using the correct elution buffer.
Antibody has not bound to immunosorbent beads	Ensure you are using the correct beads for the antibody isotype used.

Co-IP NotSuccessful

Possible Cause	Recommended Solution
Interacting protein is not present	Conduct WB to control whether interaction partner is expressed.
Protein-protein interaction has been disrupted during freezing of cells	Use fresh cells if possible. Avoid frozen cells.
Lysis buffer components or concentrations are too stringent and disrupt or inhibit protein-protein interaction	Try different binding conditions by testing various detergent and salt concentrations suitable for the protein-protein interaction. For Co-IP of soluble proteins, use a non-detergent, low-salt lysis buffer. For less soluble protein complexes, add non-ionic detergents such as NP-40 or Triton X-100 to lysis buffer.
Additives or a ligand needed for protein-protein interaction missing	Add additives or a ligand to binding buffer in order to facilitate protein-protein interaction.
Incubation time too long	Shorten incubation time because some interactions/protein complexes are only transient or unstable. A cross-linking step is required If you want to detect low-affinity or transient interactions.
Washing buffer components or concentrations are too stringent and disrupt or inhibit protein-protein interaction	Use less stringent wash buffer conditions and reduce number of wash steps. Each protein complex requires its own wash buffer composition for successful Co-IP. Save the used wash buffer from each washing step to track if the protein of interest and its interacting partners were depleted by washing.