The antibody is not capable of immunoprecipitation | Try a different antibody, polyclonal antibodies generally perform better than monoclonal antibodies. |
Not enough primary antibody is used | Determine the optimal concentration of primary antibody by titration |
There are too many competing proteins in the sample | Enrich the sample for the protein of interest, spin the lysate at 100,000g for 30 minutes before adding the antibody to remove insoluble proteins, membrane fragments, etc. |
The antigen of interest is not present | Make sure you use the appropriate sample. |
The antigen of interest is lost or denatured in the sample | Use the appropriate protease inhibitors. Prepare fresh lysates, avoid using frozen lysates |
The washes are too stringent | Reduce the number of washes. Reduce salt and detergent concentration or use a different detergent. |
There are interfering substances present in the sample | Avoid lysates containing reducing agents (dithiothreitol (DTT), 2-mercaptoethanol or other), they may destroy antibody function. Extremes in pH and excessive detergent concentrations may also interfere with the antibody-antigen interaction. |
The antibody binds weakly to either agarose beads | Use bridging antibody to enhance immunocomplex capture. |
The incubation times are inadequate | The incubation times should be appropriate for the system. Optimize the incubation times, generally, the primary antibody and antigen of interest are incubated 1 hour to overnight at 2ºC- 8ºC. |
The sample has degraded by proteases | Include additional protease inhibitors in the lysis and wash buffers. Keep the sample cold at all times. |
The antibody concentration is too low | Increase the concentration of the precipitating antibody. |
The concentration of the protein of interest is too low | Increase the precipitating antibody concentration. Increase the cell lysate concentration. Metabolically label cellular proteins. |
The antibody has low affinity for tagged protein | Use lower stringency wash buffers (for instance 150 mM NaCl with no detergent). |
The tag sequence is not accessible to the precipitating antibody, this is due to conformation of the tagged gene protein | Use alternative insertion sites within the target protein for the tag sequence. To increase the avidity of antibody reaction, insert multiple tag sequences. |
The antibody or protein incubation time is too short | Incubate with precipitating antibody for several hours at 4ºC. Incubate with protein (G/A)-agarose overnight |
The target protein is not expressed in sample used Low level of target protein expression in sample | Check the expression profile of the target protein to ensure it will be expressed in the cells of your samples. Increase the amount of lysate used when there is low level of target protein expression. However, this may result in increased non-specific binding, so it is advisable to preclear the lysate before commencing with the IP procedure. |
The target protein has not eluted from the beads | Ensure the correct elution buffer is used and that it is at the correct strength and pH for elution of the protein. |
The antibody has not bound to the immune adsorbent beads | Ensure you are using the correct beads for the antibody isotype used. |
An incorrect lysis buffer is used | Check the datasheet to see if the antibody detects the denatured or native protein and ensure the correct lysis buffer is used. |