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Insight & Intelligence™: Jun 29, 2015

Seven Tips for Choosing the Right Antibody for Western Blotting

Be Aware of the Pitfalls of Incomplete Antibody Validation Data

Seven Tips for Choosing the Right Antibody for Western Blotting

PrecisionAb mouse anti-Cdk7 antibody tested on 12 whole cell lysates. Available with positive control lysate or as stand-alone antibody.

    Ever had an antibody negatively impact your experimental results? You’re not alone. Life science is littered with the rude awakenings of researchers who watched promising results turn to dust because of a bad antibody (Baker M, 2015). In one case, results could no longer be duplicated with a new lot of antibody. In another, a researcher discovered the antibody wouldn’t bind its target protein. Projects shuttered. Time was lost, and money was wasted. It can happen to anybody. But following these rules of thumb can help ensure the antibodies you buy perform predictably.

    1. Buy for your application.  Antibodies are an incredibly versatile tool for scientists, but they have their limitations. Some are not effective at binding denatured proteins, which makes them a poor choice for a western blot. The product you buy should have been tested specifically for western blotting, with data to back that up. Be careful if there are just a few peer-reviewed articles, as it’s typical to show only the band of interest and it’s not clear what other, nonspecific, bands that may be there.
    2. Look at all available data, starting with the vendor for primary data.  Check that the antibodies cited in a published article corroborate with the applications and instructions provided on the vendor’s website. Researchers who buy products based on incomplete data risk confirming incorrect findings in the scientific literature.
    3. Be aware that not all validation is equal. Seeking vendors that are comprehensive and transparent with their studies will also give you a window into how well they validate. To save time and costs, many companies don’t test every batch, potentially releasing lots with considerable variability. Other vendors use overexpressed proteins in their studies as a shortcut to showing that their antibody binds the target of interest. The resulting data are not a good indication of how the antibody will work on an average western blot, let alone on a difficult sample. Order from vendors who validate their antibodies with whole-cell lysates, or use a range of samples, and who test every batch. (Figure above)
    4. Use third-party resources. To fast-track the referral process many organizations have been established to independently pull together data about antibody performance. Some, like CiteAb, provide a database of antibody citations in peer-reviewed publications. Other groups perform their own tests and make those data available. While they bring a lot of information together, it is often from a single type of source, most often secondary. It’s therefore important to check that the information is consistent with primary data from the vendor’s website.
    5. Consider the availability of trial-size vials. Many companies now offer small amounts of antibodies to allow you to test their product before committing to a large order. This is a good way to confirm that the antibody will work for you without risking significant amounts of money.
    6. Be prepared to optimize even a validated antibody. Vendor validation may not be able to completely replace in-house testing. Your sample may not be the same, and your protocol may be different. The combination of these and other variables will impact how the antibody performs and set your western blot apart from the vendor’s validation. You are the ultimate gatekeeper for reliability in your study.
    7. The ultimate rule. Whether you are sourcing, using, or reviewing work with antibodies, the most important thing to remember is to be aware of the pitfalls of the antibody techniques and the variations associated with them. Be rigorous when screening antibodies. Use positive and negative controls when possible and try to control your own biases to make sure your western blots provide the correct answers to your questions. 

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