Jeffrey Perkel has been a scientific writer and editor since 2000. He holds a PhD in Cell and Molecular Biology from the University of Pennsylvania, and did postdoctoral work at the University of Pennsylvania and at Harvard Medical School.
The key to a good Western blot is the antibody, and of those there is no shortage. Biocompare.com indexes close to two million in its antibody search tool, and Antibodypedia lists almost 1.2 million.
Still, sometimes the right antibody simply isn’t available. Maybe you’ve discovered a new protein or a new post-translational modification. Perhaps you’re working with a nonstandard model system. In those situations even the most extensive antibody portfolios are of little use—you’ll need to either outsource the reagent to a core facility or contract research organization (CRO), or make it yourself.
The process is theoretically simple, but tricky in practice: Antibody generation is about much more than injecting bunnies. Fortunately, there are plenty of resources to help. Here we review some key considerations.
Design your antigen
If antibody selection is key to a successful Western, antigen design is key to a good antibody. Assays like flow cytometry and immunohistochemistry (IHC) generally work best with antibodies that recognize conformational epitopes in intact proteins, because these methods interrogate folded proteins in a cellular context. In Western blots, antibodies must recognize proteins in their denatured state. Thus, the ideal antibody is one targeting a linear epitope—that is, a consecutive string of amino acids in the protein’s primary sequence.
Thus, if you’re interested only in Western blotting, a peptide antigen may be best, says Neal Kitchen, immunoassays product manager at Thermo Fisher Scientific—but only if you aren’t planning to apply the antibody to other methods. “Generally, the whole protein as antigen is most useful when you have additional applications besides Western blotting [in mind].”
But, which peptide should you choose? You need a region that is relatively antigenic, or you won’t raise an antibody response. But you also need it to be specific for your target, and that can vary with application. Sometimes, for instance, proteins are part of extended families. If you plan to use your antibody on protein lysates in which multiple family members are present, you’ll need to design your antigen appropriately. Similarly, if you wish the antibody to recognize the same protein across multiple species, you’ll need to identify regions of homology that are evolutionarily conserved.
Design your antibody
Antibody preparations come basically in two forms, monoclonal and polyclonal. (Other antibody flavors exist, such as Fab and single-chain variable (scFv) fragments, but these are more specialized.)
Polyclonal preparations are basically the serum of an antigen-injected animal, which contains many different antibodies targeting the same molecule. Polyclonals are less expensive and faster to obtain than monoclonals, but they are neither renewable (after you run out of the sera, it’s gone) nor consistent from batch to batch. Monoclonals—the products of antibody-expressing cell lines—are more expensive and take longer to produce, but they’re inexhaustible: Just thaw the hybridoma cell line and prepare another lot.
The antibody core facility at the Fred Hutchinson Cancer Research Center in Seattle charges extramural labs $13,000 for monoclonals, says manager Benjamin Hoffstrom. But that price includes anywhere from one to five antigens, so the per-antibody price ranges from $2,600 to $13,000. Turnaround time is four and a half to five months. In contrast, Abbiotec’s website lists a polyclonal cost of $1,000 to $1,500 and a three-month turnaround, and others offer them for even less.
According to Kitchen, monoclonals tend to be highly specific, and they are particularly useful when targeting specific post-translational modifications. That’s because a monoclonal is, as its name implies, a single clone—a collection of identical antibody molecules all targeting the same epitope. On the other hand, a polyclonal “tends to be more robust, in that it has multiple domains it can bind, because there are different antibodies that recognize the antigen differently.” Thus, they work better in assays, such as IHC and immunoprecipitation, in which individual epitopes can be masked by associated proteins or chemical treatments.
According to David Chimento, assistant laboratory director at Rockland Immunochemicals, the choice between monoclonal and polyclonal is in some sense academic if Western blotting is your only application—both forms should work. That said, monoclonals also typically offer lower background and higher sensitivity. “If I had to make a recommendation, and if a good monoclonal is available and not priced too high, I would go for the monoclonal,” he says.
Purify your antibody
If you opt for producing a custom polyclonal, the deliverable you receive typically is a blood-serum preparation, and you’ll probably need to do some enrichment to make it useful. At a minimum, says Chimento, do a protein A/G purification. That will isolate the immunoglobulins in the sera. But a better option, he says, is to affinity purify, using the immunogen to isolate target-specific antibodies – though he adds that such an approach is sometimes cost-prohibitive for recombinant protein antigens given the quantity of material required.
“We call that [a] ‘mono-specific’ [antibody],” he says. “It’s polyclonal, but all the antibody molecules in that product have the same specificity for the target. There’s nothing else in there.”
Mono-specific antibodies, Chimento says, work particularly well in assays like IHC, where fixation can mask specific epitopes. “Monoclonals target one epitope. If that epitope doesn’t show up, the signal won’t be visible.”
Validate your antibody
After you have your antibody, you need to make sure it works in your assay. In fact, says Craig Thompson, vice president of development and production at Cell Signaling Technology, users should also validate antibodies they buy commercially, even if the vendor demonstrates that the reagent works in the hands of its employees.
Reputable antibody vendors provide extensive datasheets demonstrating the specific protocols used to test the antibodies, effective dilutions and controls (see, e.g., this one [PDF] from CST). “If the product is claimed to work in an assay, there should be relevant assay-specific data,” Chimento says.
Even so, “every system is different,” Thompson explains. “We try to use the most relevant biological systems, but how researchers use them may vary. So it’s incumbent on the customer to do their own positive and negative controls.”
At a minimum, Thompson says, users should establish that an antibody detects what they expect to find in positive-control extracts, and nothing in negative-control extracts. But, since a band on a Western—even one of the proper size—may represent a nonspecific protein, try to use treatments that will up- or down-regulate the protein, such as drug treatments that enhance expression or siRNAs that knock it down, to ensure that you really are detecting what you think you are.
“Think of it as an experiment unto itself,” Thompson explains. “If you have to convince yourself the antibody is specific, what do you need to do?”