The Training Bench: Workout Your Immunoprecipitation Fitness Goals

Immunoprecipitation (IP) is an important assay for confirming protein-protein interactions but getting publication-quality results can sometimes be tricky. As scientists, just like weightlifters in a gym or runners on a track, sometimes we need to brush up on technique to get the best results. If your IP technique is something that you are trying to improve, this blog, can help you learn techniques to enhance your immunoprecipitation fitness!

Whether nonspecific bands are leaving you reaching for the punch bag, or the thought of troubleshooting your experiments is more daunting than a 6 a.m. gym class, you can follow our tips to help you workout your issues!

Are You Using the Right Equipment to Get Results?

In the gym, selecting the right equipment is crucial for achieving your goals. Weights are great for building strength, but less helpful if you want to learn to sprint! Similarly, in the lab, you need to choose equipment carefully to achieve your goals.

Ab Crunch: Use the Right Antibodies

Just like a piece of gym equipment, an antibody can be great for one outcome but suboptimal for another, so you should check that your antibody is suitable for use in IP. In contrast to other applications, antibodies for IP need to recognize the native form of the protein. It is no good using an antibody that only detects the denatured form, as different epitopes will be accessible. Check the datasheet to see if IP is listed as a recommended application, if it isn’t then choose a different antibody which recognizes your target and is suitable for IP.

If you are first establishing an IP protocol, polyclonal antibodies are an ideal choice as they recognize multiple epitopes and increase the likelihood of detecting an epitope that isn’t buried in the middle of the protein.

Protein Shake-Up: Use the Right Beads and Buffers

Choosing the right protein is important after a workout, and it’s important when planning experiments too (just don’t eat the science…)! In the lab, your choice of beads will impact your success. Beads are available in Protein A and G formats but antibodies have different binding affinities for these, depending on the host species and isotype. While selecting the wrong type of beads may have a minimal impact in some situations, in others it can be major.  For example, rat Ig2a does not bind to Protein A but has a strong affinity for Protein G.

Review your lysis buffer choice. You need to preserve the native conformation of the protein while still achieving efficient lysis. While radioimmunoprecipitation assay (RIPA) buffer is often a good choice, the sodium dodecyl sulfate (SDS) contained in RIPA buffer has been reported to denature kinases. Don’t forget to include protease, phosphatase, and deubiquitinase inhibitors to the lysates to preserve posttranslational modifications that may be important for interactions!

Is Your Technique Affecting Performance?

Whether you’ve been doing the same technique for a long time, or it’s bicep curls, squats, or immunoprecipitation, it can be easy to fall into bad habits. Thankfully, like you can improve technique for your push-ups in the gym, you can also improve technique for your IP pull-downs in the lab!

When performing IP, an important quality control step is to include a sample of the lysate (also known as input), alongside your IP samples when you run them on an SDS-PAGE gel. This allows you to check for the presence of your protein of interest in your lysate and confirm that the antibody used for western blot detection of your protein of interest can detect your target. If you observe a band of the correct molecular weight in your lysate, but not in your IP samples, this tells you there may be an issue with the IP procedure itself. For example, the antibody you are using for pull-down does not bind the target protein, or there was inefficient elution from the beads in the final step of the procedure.

This control allows you to compare the amount of your target protein in your IP sample, versus how much was in the lysate to perform the IP in the first place. This provides you with information about the enrichment level of your target protein.

Make Your Results Count

When training our bodies, we often want to make our training as effective as possible. The same goes for lab data, you need to make sure your results are as impactful as possible. One way to do this is to include the right controls for your experiment. Negative controls, otherwise known as an isotype control or mock IP, should be included in every IP experiment, whenever possible. Use an unrelated antibody from the same host species and isotype to the antibody used for pull-down. If you do get pull-down and see bands, it tells you that there is nonspecific binding of the beads to protein in your sample.

Fitness Tips for IP Western Detection

Below are our top tips to generate clean blots, when performing IP experiments and subsequent detection of your target protein by western blot.

1)  Remember to warm up: perform a preclearing step

Just like remembering to warm up before a run, you need to remember to perform a preclearing step before an IP experiment. This involves the addition of an irrelevant antibody of the same isotype as the IP antibody to the lysate, prior to addition of the IP antibody. The lysate which has been incubated with the irrelevant antibody is then incubated with Protein A or Protein G beads, depending on the isotype. The sample is then spun in the case of agarose and Sepharose beads, or magnetized in the case of magnetic beads, and the supernatant is removed from the beads and retained for performing your actual IP, using the antibody targeting your protein of interest. This helps to reduce nonspecific binding, as any proteins in the lysate that would bind nonspecifically to the IP antibody, due to its isotype, have been retained on the beads used in the preclearing step, thereby removing them from the lysate that you will use for the actual IP.

2) Select the level that works for you: titrate your IP antibody

In the gym, we get best results with a barbell that isn’t too heavy or too light, and we can test different weights before selecting the right one. In the lab, it’s important to select the right antibody concentration. If the concentration is too low, you may fail to immunoprecipitate your target protein, while excess antibody can lead to nonspecific binding. How efficiently an antibody binds to its target protein can vary drastically and have a massive effect on how much of your protein of interest you are able to pull down. Perform antibody titration experiments in order to determine the optimal antibody concentration to add to your beads to pull down your protein of interest. This means the lowest amount of antibody that pulls down the largest amount of your target protein and will mitigate the risk of background staining.

3) Don’t forget to shower: optimize your wash steps

Forgetting to shower after a gym session might make you unpopular on the bus home, and forgetting to wash your IP samples will affect your experiments. It might sound obvious, but failure to wash your beads sufficiently can result in the presence of nonspecific bands in your IP, leading to false positives when it comes to potential novel interactors of your target protein. Ensure that you take off as much supernatant as possible during washing without disturbing the beads. This is easier with magnetic beads, as the risk of disturbing them is lower. Increasing the number of wash steps and optimizing your wash buffer may also help to reduce background.

4) Mix up your routine: perform western blot analysis of your protein of interest using a different antibody from the IP antibody

Using a different antibody for western blot detection to the one used for IP provides assurance that the protein detected by western blotting is indeed the protein of interest, and not another protein that the IP antibody nonspecifically binds to. If an antibody is binding nonspecifically to a protein in IP, it may also bind to the same protein nonspecifically in western blotting. Ideally, you should use IP and western blotting antibodies from different host species.

You might have started off knowing squat, but hopefully this guide has helped you crunch your IP technique and left you looking forward to sit up at your lab bench for your next experiments!