Problem: Poor quality antibodies are a major stumbling block for scientists detecting new protein targets in immunological applications. The prevalence of such antibodies is significant; reactions to recent, high-profile retractions of several papers containing disingenuous Western data support this. Judging by responses to these events, it is evident most scientists have crossed paths with an inferior antibody at some point. A perceptible hint of distrust lingers over these reagents, and understandably so–immunological applications such as Western blotting and immunohistochemistry (IHC) can fail simply because of a substandard antibody. Direct consequences usually take the form of non-specific bands and staining patterns, but the real damage is often done to scientists’ time and resources.
Not all antibodies, however, are created the same. There are plenty of high-quality antibodies out there, in addition to companies that care about producing ones that work. Steps can and should be taken, where appropriate, to avoid inferior antibodies and eliminate them from the selection process before they make it to your bench.
Solution: In addition to your usual literature search, use antibody comparison sites and search engines when sourcing antibodies to fit new scenarios in the lab. Comparison sites allow you to weigh up cost and application data of multiple vendors in one place, and search engines maximize your results. Cross-reference the literature for products that have a good track record in publications.
When you begin your search keep in mind your basic criteria: which application is your top priority? Is a polyclonal or monoclonal antibody better suited? Where is your protein or epitope of interest located? Is it subject to post translational modification? Being clear on the answers to such questions will help you conduct your antibody search more objectively.
When you’ve identified a potential antibody, look at the validation data provided by the vendor; check depth and quality. Is there simply a verification of antigen recognition by ELISA testing or are there Western and IHC data too? (The more validations, the better.) Check the types of samples used for validation. Make sure you’re viewing data obtained with whole tissue or cell lysates as opposed to those prepared with purified recombinant proteins–the latter are not accurate representations of antibody performance.
If your only options lie in under-evaluated antibodies you can still find a diamond among the antibody rough–and in a risk-free environment with the right company. Look for vendors offering trial samples or covering their products with a money-back guarantee. (With the latter you can try larger volumes, still confident that you’re not wasting any of your grant money.) Guarantees should be ‘no-quibble’ and the best ones will offer the option of your full money back as opposed to credit.
Seeking out companies producing their own antibodies, as well as the larger antibody ‘supermarkets,’ has its merits. The former know their products inside and out, and can detail everything about an antibody’s production process. They can often perform validations at your request and can send you hard copies of their original validation data. These companies are also more likely to offer comprehensive and guaranteed custom production services in the event your search for a reliable pre-made antibody is unsuccessful.
For more information, visit http://whyitworks.ptglab.com/
Western blot validations hanging up in the Proteintech lab.
Correction Notice: Please note that the incorrect link appeared in the print, PDF and digital versions of this article in our October 2012 issue. The link has been corrected in this online version and the incorrect link in the digital edition has been re-routed to the proper URL.