| Target protein is not expressed in cell or tissue sample | Check literature or other resources to confirm that protein is expressed in cell type of interest Use an alternative method to confirm expression, such as IHC or ELISA Include positive control to ensure that IP is working correctly |
| Protein degraded during preparation | Add protease and phosphatase inhibitors to lysis buffer immediately before use Perform all steps on ice or at 4°C |
| Suboptimal lysis buffer was used | Try an alternative lysis buffer. Use the least stringent lysis buffer that gives acceptable yield |
| Interfering substances present in lysate | Lysates that contain dithiothreitol, β-mercaptoethanol or other reducing agents can impair antibody function |
| Insufficient antibody concentration for capture of target protein | Optimize antibody concentration by titration |
| Antibody has not been immobilized to the beads | Ensure that the bead immobilization method (e.g. Protein G) is compatible with antibody isotype - see our full guide on Immobilizing Antibodies to Beads for more detail. |
| Covalent bonding of antibody to beads or Protein A/G is interfering with capture of antigen | Use standard IP approach of Protein A/G Ig immobilization Covalently bond antibody to beads instead of to Protein A/G |
| Antibody is not suitable for IP or IP conditions | Modify conditions (e.g. use a non-denaturing lysis buffer instead of denaturing) Try an alternative antibody, e.g. a polyclonal antibody Ensure antibody has been validated for IP If using a polyclonal antibody, ensure that it has been affinity purified, as otherwise other immunoglobulins in the antisera will compete for Protein A/G binding |
| Off-target protein is successfully competing with target | Pre-clear the lysate before IP to reduce non-specific binding Try an alternative antibody with higher affinity or that recognizes a different epitope |
| Insufficient incubation to allow antigen capture | Increase incubation times and incubate at 4ºC instead of room temperature |
| Washes were too stringent or harsh | Optimize the stringency of washes by altering the salt or detergent concentration Try an alternative antibody with higher affinity |
| Protein was not eluted from the beads | Check elution buffer composition and pH Try an alternative elution buffer If mild elution buffer is suspected of being the issue, test whether protein is being sufficiently precipitated by eluting in a denaturing SDS buffer and running SDS-PAGE/western blot |
