The Western blot is the dominant means to determine the relative size (molecular weight) and abundance of specific proteins in a sample, and the basic protocol remains much as it has been for the past four decades.
That is, with the exception of how those proteins are to be detected.
From its origins of radioactively labeled antibodies exposing x-ray film, there are now a variety of ways to visualize the membrane-immobilized proteins. Which is preferred may depend on factors such as equipment availability, protein abundance, sensitivity and dynamic range desired, multiplexing requirements, and (perhaps most importantly) the need for quantification. Here we look at the advantages and disadvantages of colorimetric, chemiluminescent, and fluorescent detection methods in membrane-based Western blotting.
Qualitative westerns
Film is the most common way to detect the bands on a Western blot. After transferring the proteins to a membrane from the polyacrylamide on which they have been electrophoretically separated, the membrane is incubated with affinity reagents (typically antibodies). But rather than these being radioactively tagged as they once were, the primary antibodies are now detected by a secondary antibody conjugated to an enzyme such as horseradish peroxidase (HRP). HRP catalyzes the release of light from chemiluminescent substrate (generally termed ECL substrate), and the light is captured on x-ray film.
“I like to be able to see with my own eyes what’s on the film,” says Laura Marlow, principle research technologist at Mayo Clinic in Jacksonville. She also finds that visualization helps the students understand problems they may encounter running Westerns.
Another way to visualize a Western blot is to use a substrate such as 3,3'-diaminobenzidine (DAB) to deposit an insoluble colored precipitate on the membrane—an inexpensive technique that Kevin Janes, associate professor of biomedical engineering, teaches to his third-year undergraduate laboratory students at the University of Virginia. Documentation requires only an office scanner, “or you might as well just take a picture with your iPhone.” Unlike ECL, you can watch the color develop before your eyes, and once the reaction is halted the signal remains relatively stable. But colorimetric Western blot detection is largely confined to teaching (and perhaps some diagnostic) environments, principally because it is able to detect only nanogram-levels of protein—comparable to Coomassie.
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Film, on the other hand, is orders of magnitude more sensitive, an easy way to capture a chemiluminescent signal on a blot, and works well if you’re strictly dealing with a qualitative type of blot. But for the purpose of quantification, “no one should be using x-ray film … mainly because of linearity—it saturates way too fast and we don’t know when the film is saturated,” explains Aldrin Gomes, associate professor of physiology and membrane biology at the University of California, Davis.
Digital chemi
The same chemiluminescent Western blots used to expose film can be imaged digitally, and in so doing the issue of saturation can largely be sidestepped while at the same time providing an electronically analyzable and archivable record. There is wider, but still only a limited, range over which the relationship between the amount of protein loaded and signal is linear, and “you have to get the enzyme kinetics just right,” notes Janes. Fortunately, “the software that comes with machines now is so good they can show you when it’s saturated,” Gomes says. A researcher will typically take multiple exposures in order to get the best image.
While the basic components of ECL reagents are standard, “different companies have different mixtures,” points out Gerry O’Beirne, global product manager, electrophoresis and Western blotting at GE HealthCare Life Sciences. There are variances in how the solutions are prepared, shipped, and stored, for example, how stable the signal is and how long it lasts.
But perhaps most importantly, several vendors now offer choices of sensitivity levels, with some less likely to saturate when probing for very abundant proteins such as actin, while at the other end are reagents that claim a lower limit of detection rivaling that of 32P. The latter are generally recommended only for low-abundance proteins— “very weak signals where you’re willing to sacrifice some of the quality, the cleanliness” of the blot, says Marlow.
On the other hand, Gomes has found that judicious use of these pricier high-sensitivity ECL reagents—even to detect high- or moderate-abundance proteins—can save money. Antibodies account for a significant proportion of the operational cost of running a Western blot, and “we’re using up to ten times less antibody.”
Fluorescence
Like an increasing number of researchers, Janes relies mostly on fluorescence detection, in which the secondary antibody is conjugated to a fluorophore (rather than an enzyme), and on “chemiluminescence detection [only] when we think we have a signal that is too weak to be detected by fluorescence.” A side-by-side comparison revealed “pretty compelling” results that fluorescence was superior to chemiluminescence at Western blot quantitation, he explains. “The numbers that you get on the fluorescence detection will be much more reliable and reproducible.”
The sensitivity of fluorescence Western blot detection rivals that of ECL, and is based on a number of factors. These include quality of the antibody, the excitation and emission spectra of the fluors, the equipment used to image the blot, and background (auto)fluorescence of the membrane. O’Beirne emphasizes that optimization of each step of the process, including antibody dilution and buffer conditions, contributes to the equation as well.
An imager capable of reading a fluorescence Western blot can be more expensive than one capable of just reading chemiluminescence (more vendors are now marketing multi-modal instruments as well). But it offers the opportunity to multiplex two, and in some cases three, different signals simultaneously from the same sample—allowing, for example, for detection of phosphorylated versus non-phosphorylated proteins “on the same blot, without stripping,” exclaims Gomes. “We cannot do that with chemi.”
“Another advantage of fluorescence is that you can take that blot and keep it for weeks to months, and it will still give you a signal, whereas with chemi it’s gone within hours, to a maximum of a day,” Gomes notes.
The bottom line
Western blotting is a versatile technique capable of delivering an inexpensive answer to whether a protein is present in a sample.
Optimization steps, learning curves, and the time to actually run the experiment are essentially the same up until the final detection schema. And even there, for all three methods are roughly equivalent. One advantage that chemiluminescence may have is that as the still-dominant method, there are more published papers, and likely more experienced colleagues, to rely upon for help.
Low set-up cost is the main—and to some researchers the only—selling point for colorimetric detection. Sensitivity to detect minute amounts of protein gives chemiluminescence an edge (although this is not uncontested)—that, and cost of equipment. And for reproducibility, quantitation and multiplexing, fluorescence clearly leads the way.
Image courtesy of Dreamstime Images.