In the corporate hierarchy of the cell, DNA holds the C-level offices while RNA staffs the middle managers. But the worker-bees that actually do the dirty work? Those are proteins, and by monitoring what those proteins are doing, researchers can work out what’s going on at cellular HQ.
Here’s the thing, though: If you want to measure a cell’s response to some stimulus or event, it’s usually not enough to measure protein abundance. Eukaryotic proteins are frequently subject to one or more post-translational modifications, from phosphorylation and methylation to acetylation and ubiquitination. And those modifications can alter protein activity, stability and location in ways both subtle and profound.
Perhaps the most widespread and useful tool for tracking post-translational changes is the modification-specific antibody. Thousands have been developed over the years—the Cell Signaling Technology (CST) catalog lists more than 1,400 modification-specific antibodies out of a total portfolio of 5,516, for instance—and researchers can use them to drive a variety of assays, including such biochemical assays as Western blotting, chromatin immunoprecipitation and flow cytometry.
For protein quantification in situ, researchers can use immunohistochemistry, immunofluorescence or DuoLink. A cross between PCR, immunofluorescence and fluorescence in situ hybridization, DuoLink uses pairs of oligonucleotide-conjugated antibodies recognizing both the protein and its modification to drive a 100- to 1,000-fold signal amplification of a given protein signal, explains Karen Kwarta, global product manager at Sigma-Aldrich, which commercializes the assay. The assay readout uses fluorescent oligo probes complementary to the amplification product, indicating not only where the modified protein is located but also its abundance. “Because this is a technique that generates this long strand of DNA that stays in place, you basically have a digital count,” Kwarta says.
Given the breadth of today’s antibody collections and the speed with which they are growing, there’s a good chance you can find one to match your protein target, whether your focus is epigenetics, cell signaling or cancer. But just because two antibodies target the same sequence, it doesn’t mean they are equal. With lab budgets tighter than ever, it’s critical to make the right choice. Here’s what to look for.
Making (and validating) an antibody
Antibodies targeting a protein (regardless of modification state) can be made using the whole molecule as an antigen—a strategy that ensures a range of epitopes will be represented in the final serum. Modification-specific antibodies, though, typically are prepared using synthetic modified peptides, says Stefan Pellenz, head of quality control at Antibodies-Online. “The problem is, you want to exclude antibodies [that] recognize nonphosphorylated forms, which is very tricky.
Antibody developers must also exclude antibodies that recognize a given modification in the context of other similar peptides or modifications, as well as those that recognize a peptide in the absence of the modification itself.
At the same time, the antibody must function in the assays for which it is intended, says Matthew Stokes, group leader of the proteomics service group at CST. Antibodies against histone modification, for instance, are most likely to be used in chromatin immunoprecipitation and are tested accordingly. But for the company’s recently released line of antibodies targeting tumor immunology proteins such as PD-L1 and VISTA, the key application area is immunohistochemistry. “For each antibody we make, we try to define ahead of time what we think the key applications will be,” Stokes says.
Site-specific antibodies may be either monoclonal or polyclonal. The latter are easier and faster to develop, but both require significant validation time, says Carolyn Crankshaw, global product manager for antibodies and assays at Sigma-Aldrich. In the case of a post-translational modification, for example, that could involve counterscreening with an unmodified peptide to reduce nonspecific binders, she says.
Particularly crucial, says Stokes, is testing antibody reactivity against well-established positive and negative controls. And that includes more than purified, recombinant protein standards, he adds: “We strive to develop antibodies that recognize endogenous protein expression, which requires model systems," such as a change in target abundance upon treatment with an activator or inhibitor, or in the presence of specific siRNAs.
A new modification
Though researchers often want antibodies that recognize a specific modification in a particular context—c-Abl phosphorylated on tyrosine-245, for instance—more broad-specificity reagents also are available, such as antibodies that recognize generic phosphotyrosine residues regardless of their peptide context.
CST has developed so-called “Motif antibodies” that recognize a range of modifications, including ubiquitination, methylation and lysine acylation. According to Stokes, researchers can combine these with additional applications to identify and quantify proteins containing a given modification (mass spectrometry), screen for global changes (Western blotting) or look for modifications to specific proteins (immunoprecipitation/Western blotting). The company also offers services such as PTMScan (mass spectrometry-based) and Kinome View (Western blot-based) for users who lack the time or experience to collect those data in-house.
Earlier this year, EMD Millipore launched a series of antibodies against another type of modification, a little-known form of phosphorylation called phosphohistidine.
Phosphohistidine, says Jeff Xu, the company’s senior product manager for signaling and immunology antibodies, was discovered some 50 years ago in bacteria. But the modification has been little investigated in eukaryotes, in part due to a lack of antibodies specific for the modification. That’s because unlike phosphoserine, for instance, phosphohistidine is highly unstable. “The difficulty in designing such an antibody is that the bond is very, very weak,” Xu explains. “So it’s very difficult to create an antigen, because the epitope simply doesn’t last long enough before it is hydrolyzed.”
In July, a research team led by Tony Hunter at the Salk Institute for Biological Studies in La Jolla, Calif., figured out a way to circumvent that problem using nonhydrolyzable chemical analogs [1]. Millipore now offers four reagents—two antibodies for each of the two forms of the modification: 1-phosphohistidine and 3-phosphohistidine. Researchers, Xu says, have long suspected a role for phosphohistidination in eukaryotic biology, but have lacked the tools to investigate that possibility. “Having the antibody available really allows the researchers to look into all kinds of different events that phosphohistidine can impact.”
Antibody validation
Whatever its target, when buying a new antibody (or even new batch of antibodies), be sure to read the product literature to understand how the antibody was tested. Is the antibody rated for your application? Does the vendor test each lot? And are the validation data compelling? "If they are just running a Western blot with recombinant protein, then I would contact the company to see if they know whether it detects endogenous protein and can provide supporting data,” Stokes says.
Then, after you have the antibody in hand (or rather, on ice), test it yourself, says Pellenz. “The most basic reagent, such as a wash buffer, can ruin the experiment if the pH is off,” he notes.
Pellenz’s “main recommendation” is to make sure you have well-validated, reliable positive and negative control samples—cell lines in which you are certain the protein is or is not expressed or modified. “Especially, negative controls can be very, very tricky. So maybe use multiple parallel controls,” he advises.
Jahan Ara, senior scientist at Rockland Immunochemicals, recommends testing antibodies against specific peptides at different concentrations on a dot blot assay or ELISA—a method that can reveal both antibody specificity and sensitivity.
Finally, if you can afford it, consider testing different batches of antibody from different vendors. Or don’t, says Xu. “Instead, invest that money in better controls.”
Reference
[1] Fuhs, SR, et al., “Monoclonal 1- and 3-phosphohistidine antibodies: New tools to study histidine phosphorylation,” Cell, 162:198-210, 2015. [PMID: 26140597]