Three-plus decades after it was invented, Western blotting is among the most widely used techniques in today’s biology labs.
Involving separating proteins by size on polyacrylamide gels, transferring (or blotting) those proteins to a membrane and then probing that membrane with specific antibodies, Western blotting affords a straightforward method for screening samples for the presence, abundance and post-translational modification of proteins of interest. But being straightforward doesn’t necessarily make it easy.
"Western blotting is (for many) a surprisingly persnickety method, requiring considerable hands-on time and optimization . "
Fortunately, vendors continue to innovate, providing a new crop of tools to ease your Western worries.
Choosing a detection method
Researchers have three options when it comes to Western blot detection. Chemiluminescence-based detection, using a horseradish peroxidase (HRP)-conjugated secondary antibody and an enzymatically activated substrate, is far and away the most common choice, says Michele Hatler, senior product manager for immunodetection products and Western blotting at MilliporeSigma. Alternatively, researchers can opt for colorimetric detection, which typically relies on alkaline phosphatase or HRP-conjugated secondary antibodies, or fluorescent detection with secondary antibodies coupled to fluorescent dyes (or, in the case of Thermo Fisher Scientific’s WesternDot™ reagents, fluorescent quantum dots).
As a general rule, says Raymond Miller, product manager for protein quantitation products at Bio-Rad Laboratories, chemiluminescence offers the greatest sensitivity—down to the femtogram level in some cases—followed by fluorescence and then colorimetry. But that sensitivity can also vary over several orders of magnitude depending on the specific detection reagent used. For instance, Thermo Fisher Scientific’s SuperSignal West Pico reagent offers picogram sensitivity, and its SuperSignal West Femto reagent can pick up low femtogram-level protein signals. In the middle of the detection scale, the company’s SuperSignal West Dura reagent provides mid-femtogram sensitivity and hours-long signal duration, says Priya Rangaraj, global market development manager for protein analysis products.
Bio-Rad’s Clarity™ Western ECL reagent also lasts up to 24-hours, according to Miller. Most chemiluminescent reagents provide relatively short-lived signals, complicating lengthy and repeated exposures.
But, users should resist the urge to automatically reach for the highest-sensitivity detection reagents, Rangaraj says. High-sensitivity reagents such as SuperSignal West Femto are intended for low-abundance or difficult-to-detect proteins; when used on high-abundance proteins, they can produce messier results.“With great sensitivity comes greater background,” she quips.
Though not as sensitive as chemiluminescence, fluorescence is generally acknowledged to be superior when it comes to quantitative Western blotting, especially given that fluorescence Western blots can be multiplexed, meaning researchers can directly compare the signal of, say, one protein to a housekeeping protein, or a specific phosphorylation event to the protein’s total abundance. Chemiluminescent and colorimetric blots cannot typically be directly multiplexed, unless the target proteins of interest are of different sizes. Instead, the blots must be stripped and reprobed, a laborious and unpredictable process.
Alternatively, researchers can use fluorescence to compare a specific protein signal to total protein loading, a superior normalization strategy vs. using housekeeping genes, says Miller. Bio-Rad’s V3 workflow, for instance, uses fluorophore-embedded precast gels. Exposing those gels to UV light crosslinks the fluorophore to the separated proteins, meaning no additional post-electrophoretic staining step is required. “And then, at every subsequent step, you can visualize what’s happening,” Miller says. Similarly, GE Healthcare Life Sciences’ Amersham™ WB System enables users to pre-label their protein samples with Cy™5, reserving the second fluorophore channel (Cy3) for detecting the protein of interest.
Although few researchers opt for colorimetric detection these days, Hatler says, companies still support it. Thermo Fisher Scientific’s new UltraTMB blotting reagent for colorimetric HRP detection, for instance, offers sensitivity on par with “entry-level” chemiluminescence detection, says Rangaraj, “but without the darkroom.”
Given the choice between chemiluminescence, fluorescence and colorimetric detection, a good starting point is chemiluminescence, especially for simple yes/no detection of reasonably abundant proteins. “If the general application is just protein identification, chemiluminescence will give you that, easily,” Hatler explains.
Simplifying and automating
Despite its ubiquity, Western blotting is a laborious, time-intensive and generally irreproducible process. But companies have developed systems and strategies to mitigate those challenges.
Bio-Rad Laboratories’ V3 (visualize, verify, validate) workflow, for instance, improves Western blotting reproducibility by enabling users to monitor their experiment at different stages of the process, says Miller. “Western blotting is a very mature technology, and it’s very much a black box,” he says. With V3, users run their samples on precast, fluorophore-embedded SDS-PAGE gels; expose those gels to UV light to crosslink dye to protein; and then transfer the labeled proteins to a membrane. Then, following incubation with primary and secondary antibodies, researchers can visualize their blots using the company’s ChemiDoc MP imager, a chemiluminescence- and fluorescence-based imaging system that includes both a UV transilluminator (for DNA gels) and RGB fluorescence.
Bio-Rad has also launched a rapid blotting system, the TransBlot Turbo, for protein transfers in as little as three minutes. “This is a real advantage over traditional wet transfers,” Miller says. And the company has recently entered the antibody market with a portfolio of some 600 PrecisionAb™ antibodies, all of which are validated for Western blotting.
GE Healthcare Life Sciences’ “flagship” Amersham WB System accelerates the Western blotting workflow down to about four and a half hours from protein loading to analyzed data, according to Martin Teichert, global product manager for the company’s Biomolecular Imaging Group.
Intended to reduce hands-on time and increase reproducibility, the Amersham WB System comprises two units, Teichert explains, one for running the gel and performing detection and another for gel transfer, washing and probing the blots. In January, the company made the gel/detection unit available as a standalone instrument called the Amersham Phast System™. According to Teichert, users load their gel with samples pre-mixed with fluorescent dye. After the system detects a mobile front, it automatically performs a complete imaging scan of the gel, no staining required.
MilliporeSigma’s SNAP i.d. 2.0 system is a vacuum-driven instrument that simplifies the Western protocol’s laborious immunodetection steps. According to Hatler, the system performs both primary and secondary antibody incubation, as well as blocking and washing steps, in just 30 minutes, providing a highly efficient workflow. “The vacuum drives the reagents through the membrane, so you don’t have to wait for gravity and diffusion.” (Typically, primary-antibody incubation alone can take from four hours to overnight.)
Thermo Fisher Scientific also is working to accelerate the Western blotting workflow with the iBind™ Flex Western Device, an automated Western blot processing system that completes the protocol’s immunodetection steps in three hours with walkaway automation. According to Rangaraj, “You can leave it overnight; it will be fine. And no power is required. It runs by [sequential] lateral flow.”
Also available from Thermo is its new SureCast™ gel-casting system. According to Rangaraj, precast gels offer convenience and reproducibility, but at a price. As a result, some researchers prefer to pour their own gels when they can. The SureCast system features thick, shatter-resistant glass plates, a simple gel-pouring apparatus, and is meant to be “100% leak-free,” Rangaraj says. The company also supplies room-temperature-stable acrylamide and polymerization reagents.
Advice for newbies
These tools undoubtedly simplify the Western blotting workflow. But they don’t remove every difficulty. In particular, all agree, be prepared to optimize the assay and conditions.
“People think there’s this rigid protocol, and all the steps are the same regardless of the protein,” says Elizabeth Smith, product manager at MilliporeSigma. But that’s not true, she says—from the choice of membrane and blocking reagent to antibody dilutions and washing conditions, every variable can influence data quality. Smart researchers optimize them for every new set of experiments.
It’s easy to blame the antibody for a lousy result, Smith says, but they’re not always at fault. (Though some companies, such as St. John’s Laboratories, allow researchers to try antibodies before they buy.) “Western blots can be finicky, and it takes time to be able to optimize them,” Smith says. “Every single reagent should be fine-tuned for whatever experiment you’re doing.”