Immunoprecipitation - Protocol & Troubleshooting

Troubleshooting

Although the IP method is very simple. However, there can be various specific differences between different proteins and different antibodies. Therefore, the variables and factors that affect the success of an experiment are not only numerous and varied. We describe some common troubleshooting tips for you to help you continuously optimize your experimental conditions and finally get satisfactory isolated proteins.

No eluted target protein detected

1. Sample causes. One possibility is that the target protein is not expressed in the sample used or is expressed at a low level. You should check the expression of the target protein. If the target protein expression level is low, you can increase the amount of lysate. But at the same time this may lead to an increase in non-specific binding. Therefore, we recommend pre-clearing the lysate before starting the IP procedure.
   Another possibility is that there are too many competing proteins in the sample. We recommend centrifugation after cell lysis to remove insoluble proteins, membrane fragments, debris, etc.
   It may also be that the target antigen is lost or destroyed in the sample. You can try preparing fresh lysates under appropriate protease inhibitors and prevent freezing.
   Finally, there may also be interfering substances present in the sample. Some lysates containing reducing agents can disrupt antibody function and should be avoided. Extreme pH values and excessive reagent concentrations may also interfere with antibody-antigen interactions.
2. Lysis buffer causes. It is possible that you used the incorrect lysis buffer. First make sure that you are using the correct lysis buffer. You can use a different lysis buffer to improve solubility. Or try to denature the lysis conditions. On the other hand, the amount of lysis buffer is not enough. You should increase the amount of lysis buffer and pre-clean the lysis buffer before starting the IP procedure.
3. Antibody causes. It is possible that you do not have enough antibody to capture the target protein. Check if using the recommended amount of antibody. Determine the optimal concentration of antibody by titration experiments. Or perhaps the antibody you selected is not suitable for immunoprecipitation. We recommend that you try different antibodies and use an antibody with a higher affinity for the target. Polyclonal antibodies usually perform better than monoclonal antibodies. Also, if the primary antibody is not fully captured, use a secondary antibody.
4. Bead causes. You may have used the wrong type of beads, resulting in antibodies not binding to the immunosorbent beads. Make sure you are using the correct type of beads. It is also possible that the antibodies are weakly bound to the beads and you can change to another brand of beads.
5. Incubation causes. Incubation time is too short. Usually, the antibody and the target antigen are incubated for 4 hours to overnight.
6. Washing causes. Too strict washing causes. Reduce the number of washes and detergent concentration. Avoid washing buffer stripping immune complexes from agarose beads.
7. Elution causes. The target protein is not eluted from the magnetic beads. You need to make sure that you are using the correct elution buffer and that it has the correct strength and pH.

High background

1. Sample causes. When preparing cell lysates, firstly avoid freezing the lysate. Next ensure to add fresh protease inhibitors to prevent antigen degradation. Too many cells can result in a large number of non-specific proteins in the eluate. You can reduce the number of cells/lysates used. As well as reducing the non-specific binding of proteins to beads or antibodies by pre-clearing the lysate if necessary.
2. Antibody causes. Too much antibody can lead to non-specific binding. We recommend determining the optimal concentration of antibody by titration. It is also possible that the antibody used is not specific enough, so use an affinity-purified antibody. Precipitation may be present in the antibody and you will need to centrifuge to remove any particulate matter.
3. Bead causes. Non-specific protein binding caused by beads that are not adequately pre-closed. Ensure that the blocking agent is fresh and in saturated amounts.
4. Washing causes. Washing is not thorough. Be sure to wash well at the relevant stage. Use a more stringent washing solution or increase the number of washes.

Other problems

1. A large number of antibodies eluted. We recommend trying to reduce the amount of antibody used. Cross-link the antibody to the beads prior to IP and elute using a mild buffer gradient.
2. Protein of interest blocked by antibody heavy or light chains. To avoid interference from the antibody chain, we recommend the use of secondary antibodies.