Histone antibody specificity has been the topic of publications as well as discussions at meetings and conferences (probably water coolers and coffeemakers, too) within scientific community recently. Clearly, antibody specificity is important to researchers, but what are vendors doing to address it? To get an idea of what goes into making better histone antibodies we spoke with Anita Lozsa, M.S. Antibody Development Scientist and Michael Sturges Ph.D., Sr. Product Manager from EMD Millipore see what they are doing to take on this challenge.
Can Histone Abs Be More Specific, Please?
As extremely sensitive, data and resource intensive, genome-wide epigenetic studies become more en vogue; the stakes continue to be raised for the antibodies that those techniques rely on. “Concern over the specificity of antibodies is nothing new it’s just become more critical. “ Says Sturges.
“Our R&D group understands the impact of antibody quality on genome wide techniques through the development of our ChIP-chip and ChIP-Seq kits. When performing ChIP analysis using microarrays and next generation sequencing it is very important to have highly specific antibodies to avoid enriching for non-specific regions that can cause misleading results.” Explains Lozsa, “Nobody wants to find after many months of work that their data was incorrect or even worse, published and in need of retraction.”
“Plus, for many labs funding is tight. Non-specific antibodies can cost labs time and money. Nobody, regardless of their level of funding wants to waste resources.” Adds Sturges. There’s no doubt about that. The need for quality antibodies has even prompted groups like the ENCODE and Roadmap Epigenomics initiatives to create an Antibody Database to record the performance of antibodies they’ve tested, and let others see the results before they try them on their own.
Connecting the Peptide Dot-blots
For it’s part, EMD Millipore has put a ton of effort into its rigorous antibody characterization process.
“We always perform immunoblots and evaluate performance in applications, such as immunoprecipitation, immunocytochemistry and chromatin immunoprecipitation. However, the correct band on a Western blot does not guarantee specificity. For example it is quite possible that an antibody against say H3K27me3, will also react with H3K27me2, the unmodified histone or even some other modification site.” Says Lozsa. “Because of this, we have tried evaluating the specificity of our histone antibodies with Luminex assays, peptide inhibition assays (PIA), and peptide dot blots (DB), but most of our screening is now done by DB.”
According to Sturges, “Using dot blots allows R&D to more rapidly and easily screen a greater variety of potentially cross reacting sites, because it’s possible to spot 96 peptides on a membrane and get a broader profile of the cross reactivity of a given antibody. There are also a couple of pieces of data suggesting that dot blots might be more sensitive than peptide inhibition assays.”
“On the rare occasions when DBs show low level cross reactivity that PIA doesn’t detect, we have included both sets of data in the product data sheet and website. We feel it is important to give as much detail on specificity as we have available.” Sturges added
Lozsa points out that for any application it is important to determine an antibody’s optimal dilution, which can be crucial to experimental success. “How much antibody is used for an application can really impact specificity. For example if you use too much, it’s going to cross react with a lot of other proteins no matter what, especially if it’s a polyclonal. On the other hand, if the dilution is too high you won’t be able to detect anything at all.”
The Rewards of Antibody Specificity Screening
So the end result of this continually evolving specificity testing is of course, better antibodies on the market. But it has also led to a refinement of the entire production process. As Sturges puts it, “Screening for specificity and looking at both successful and unsuccessful strategies, we have gained insights across the entire antibody development process – everything from immunogen design through final formulation has benefited. “
“We continuously learn something new about the nature of the antibodies and improve our testing and validation process accordingly.” Adds Lozsa.
“If I were running time consuming and expensive applications like ChIP-seq or ChIP-chip, that generate a lot of data, I would want to make sure I had the best performing, best characterized antibody available.” Says Sturges. Which is why the EMD Millipore team feels like the extra work that they put into antibody specificity screening pays off in a big way.
You can check out the entire line of antibodies at the EMD Millipore website.