Caitlin Smith has a B.A. in biology from Reed College, a Ph.D. in neuroscience from Yale University, and completed postdoctoral work at the Vollum Institute.
Though Western blotting has for years been an important mainstay for protein researchers, many believe it has yet to reach its full potential. Performing Westerns has gradually become more convenient for lab workers, and easier for novices to do successfully. Recently, the field has seen dramatic advances in ease-of-use, many of them addressing an essential quandary – researchers are often forced to make a choice between speed and sensitivity. And protein quantification remains tricky.
Meeting these challenges is easier with recently developed Western blotting tools. “Researchers are looking for any product that enables them to perform Western blots more quickly without sacrificing quality or sensitivity,” says Priya Rangaraj, market segment manager for Thermo Scientific Protein Detection at Thermo Fisher Scientific. Indeed, some systems offer completed Westerns in minutes or hours rather than the traditional days-long protocols. “I believe the industry will continue to try to shave time off this process, but researchers will always need to maintain – or even improve – the sensitivity they’ve come to expect from this important protein detection method,” says Rangaraj.
Whatever the speed, Western blotting is a process comprised of many variables and steps, any of which could go awry and compromise results. For a reliable Western blot, “sensitivity of detection reagents, buffer compositions, blocking agents, antibody specificity and incubation times all have to work successfully,” says Michele Hatler, product manager in Western blotting solutions at EMD Millipore. Researchers are getting a handle on the problem spots, and trying to anticipate future challenges, using recent advances in Western blotting tools.
Speed without sacrificing quality
One impressive advance is speed. Speed and quality of Western blots don’t have to be mutually exclusive anymore. For example, Thermo Fisher Scientific’s Fast Western Blot workflow, a series of reagents that shave the time after the running the gel down to about an hour. “It takes a blot from blocking to detection in minutes versus hours,” says Rangaraj. They also recently launched a series of new blocker and buffer reagents compatible with all types of Western blots.
Likewise, EMD Millipore offers a new fast western blotting system, the SNAP i.d.® 2.0, which completes the immunodetection steps of a blot in 30 minutes using vacuum force. “Unlike conventional Western blotting, where diffusion is the primary means of reagent transport, the SNAP i.d. 2.0 system applies a vacuum to actively drive antibodies and buffers through the membrane,” says Hatler.
Bio-Rad’s V3 Western Workflow offers fast Western blotting with built-in checkpoints to ensure the blot is running with the expected quality. The V3 Western Workflow consists of their Trans-Blot Turbo blotting system, Stain-Free technology and precast gels, and their ChemiDoc MP imaging system. After a 15-minute electrophoresis run, the gel is imaged for 1 minute to confirm good separation, and blotted to a membrane in 7 minutes with the Trans-Blot Turbo. “Due to Stain-Free technology, the membrane can then be visualized in under a minute to confirm a high quality transfer,” says Ryan Short, Western blotting marketing manager at Bio-Rad Laboratories. “The V3 workflow adds the confidence of knowing that [the electrophoresis and blotting] steps went well before starting expensive and time-consuming antibody incubation steps.” Short says that half the researchers polled recently said their Western blots “fail at least 25% of the time. We also know that for most people Western blotting is a two day process. We believe that it does not have to be so inconsistent or slow.”
High-quality antibodies can make a big difference in the quality of your results, yet finding the right antibody for your experiment is not always simple. “One of the biggest challenges researchers have with Western blotting is finding high-quality, validated antibodies because they ultimately determine the quality of the blot,” says Rangaraj. “A bad antibody means you’ll end up with a ‘dirty blot,’ even if you use good reagents.” To help researchers wade through the many thousands of antibodies available today, Western blotting suppliers are selling pre-validated antibodies that have been tested on their blotting systems, and shown to work well under common-used conditions. Rockland Immunochemicals takes this validation further by testing antibodies at all stages of production to keep tabs on consistency between lots, which helps to minimize variations in the data.
Enhancing signal detection
Antibodies are also commonly used prior to Western blotting in immunoprecipitation studies, followed by Western blots to verify the identities of precipitated proteins. Rockland Immunochemicals offers the TrueBlot® immunoprecipitation and Western blotting system, which is designed to enhance the detection of target protein(s), without the signal of the immunoprecipitating antibody interfering. “Peroxidase-conjugated TrueBlot® reagents accomplish this by preferentially detecting the native form of the primary antibody,” says Carl Ascoli, laboratory director at Rockland Immunochemicals. “TrueBlot® is a unique secondary antibody for western blot detection of immunoblotted target protein bands, without interference from the 55 kDa heavy and 25 kDa light chains of the immunoprecipitation antibody that may mask proteins of interest.”
Other tools for enhancing detection include reagents that assist in distinguishing signals of interest from background noise. EMD Millipore’s Bløk Noise Cancelling Reagents reduce the background noise caused by non-specific antibody binding “without leaving a thick sticky layer like milk,” says Hatler. “Bløk is the only protein-free, noise-canceling reagent available, which means you can stain after immunoblotting.” In addition, their Signal Boost™ Immunoreaction Enhancer, when used during incubations of primary and secondary antibodies, “increases the binding efficiency of the antibodies to their target epitopes, hence increasing the signal on a Western blot,” says Hatler. As a result, previously undetectable protein bands may be revealed, and already detectable bands may require less antibody.
While Western blotting is excellent for confirming the presence of proteins, its ability to quantify proteins remains challenging. Typically, researchers normalize protein expression levels using the relative expression of their target protein and a common housekeeping protein. However, different experimental conditions can lead to changes in expression levels of those housekeeping proteins. “Furthermore, housekeeping proteins are often highly expressed whereas the proteins being studied are expressed at very low levels,” says Short. “This makes it difficult to load enough protein into each well and still quantitate both the housekeeping and protein of interest within the linear dynamic range of the assay.” Bio-Rad’s V3 Western Workflow aims to circumvent this issue by using total protein normalization, in which the signals of target bands are normalized to the signals of all the proteins in the same lane. With Bio-Rad’s Stain-Free technology, “this intensity is easily captured from the blot in a single imaging step that takes less than a minute,” says Short. “Compare this to commonly used methods like stripping and re-probing the blot for housekeeping proteins, which can take hours.” Bio-Rad also has a new reagent, the Clarity Western ECL Substrate, that is designed to provide researchers with low-cost, yet high-performance and bright signals.
Digital Westerns
Though film is still widely-used for recording chemiluminescent signals on Western blots, researchers may increasingly turn to digitization in the coming years. The advantages of digitally imaging your Western blot include faster results that can be stored digitally and do not degrade over time, and no need to handle hazardous chemicals that accompany film development. Sometimes it isn’t a choice - some labs simply don’t have or can’t afford a darkroom or (traditional) imager.
Thermo Fisher Scientific’s new Thermo Scientific 1-Step Ultra TMB-Blotting Solution is a substrate with sensitivity similar to that of chemiluminescent substrates, and “is ideal for labs that don’t have a darkroom or imager,” says Rangaraj. Their Thermo Scientific myECL Imager can capture and analyze signals from chemiluminescent and colorimetric substrates without a darkroom or film.
Similarly, LI-COR Biosciences’ new C-DiGit Blot Scanner for chemiluminescence-based Western blotting is designed to make Western blotting accessible to more researchers by eliminating the need for film to image chemiluminescence signals. “We wanted to offer a sensitive, yet affordable system to everyone working chemiluminescence-based Western blots,” says Jon Anderson, senior scientist at LI-COR Biosciences. “The C-DiGit is a personal-sized imager that provides the sensitivity and image quality that film users are accustomed to seeing.” Despite being the size of a lab notebook, this imager can capture an entire Western blot in one scan. “Just as digital cameras replaced film-based cameras, we see the C-DiGit System as a legitimate replacement for film-based chemiluminescent Western blotting,” says Anderson.
Digital Western blots may also help researchers wrestle with protein quantification. “Film-based chemiluminescence detection is currently the most widely-used detection technique, yet it is the most challenging technique for accurate quantitation,” says Anderson. “Limitations due to enzyme and substrate kinetics, signal instability, signal saturation, time-dependent detection, and non-linearity of film make accurate quantitation extremely difficult.”
In addition to quantification, cost is another reason to consider digital Westerns. As medical imaging increasingly uses digital methods, the cost of film is rising and its production declining, according to Anderson. Thus researchers who depend on film for Western blotting may soon encounter problems obtaining film, and would be well-served by the availability of economical digital imaging technology. “The entire field of digital imaging is making great advances over film,” he says. “The ability to image, analyze, and store digital Westerns is what’s needed by researchers.”
As with other areas of science, automation is making an entrance into western blotting too. Protein Simple has made Western blotting more hands-free than ever before. After loading a sample into one of their automated blotting systems, the human has no further role in the technique until it’s completed. Protein Simple’s instruments automate sample loading, protein separation by size or charge, antibody incubations, washes, chemiluminescent signal detection, and even data analysis. An instrument can process up to 96 samples simultaneously, and a complete run takes 19 hours. The higher throughput of Protein Simple’s systems are due to their use of capillary electrophoresis, where proteins are separated in tiny fluid-filled tubes, rather than traditional slab gels. The results can be displayed as an electropherogram – a capillary electrophoresis readout – or as a mock-blot that converts results to lane format. For small, precious samples, they also offer the automated NanoPro 1000 system, which can separate proteins by charge using a sample of only 25 cells.
Researchers who rely on Western blotting undoubtedly appreciate its robust and consistent ability to detect protections – but they continue to demand more from the technology, such as “tools to detect multiple proteins within the same sample, modifications associated with those proteins, multiple post-translational modifications of the same protein, increased throughput of Western blots and the ability to maximize the amount of data from a single gel,” says Hatler. “[This] continues to drive improving sensitivity and speed of blotting techniques.”