Editorial Article
Monday November 03, 2008
by Caitlin Smith
If you think you know all about Western blotting, read on. The latest technological developments for Western blotting have made it useful in areas where once it did not tread.
“Western blotting is an inherently qualitative technique that has withstood the test of time,” says Samesh Kale, product manager in the bioscience division at Millipore. “Nonetheless, given the increasing experimental sophistication of protein research, scientists are looking to bring reliable, consistent quantitation into their labs and are adapting Western blotting to this end. I see fluorescent Western blotting as one of the key tools used by scientists to bring a level of quantitation to protein research.” Westerns still can be too time-consuming or unreliable. Despite these caveats, we are changing Western blotting in ways that expand its previous limits.
Faster answers
The latest Western blotting kits now give researchers the option of reducing their Western blot detection times to one hour or less. For example, GenScript’s new One-Step Western™ Kits are meant to cut blotting time from 4 - 5 hours down to one hour without sacrificing sensitivity. Their One-Step Western™ Fluorescent Kit, which uses fluorescently labeled secondary antibodies, “is optimized and more sensitive than traditional chemiluminescence methods,” says Jason Zhou, marketing specialist at GenScript. In addition, their One-Step IP-Western™ Kit “not only trims IP-Western analysis time to one hour, but also completely eliminates all contamination from heavy chains, light chains, and proteins A and G,” says Zhou. “Compared with competitors’ products, we found the competitors cannot totally block Protein A and G contamination, and their kits cannot be used for all mouse species.”
Millipore also aims to accelerate the detection phase of Westerns with their new SNAP i.d. Protein Detection System, which uses vacuum transport through the blotting membrane. Kale says that the system reduces immunodetection to 30 minutes. “With the SNAP i.d. system, researchers can begin their Western blots in the morning and have their results to analyze by lunch,” says Kale. “SNAP i.d. is the only product on the market today that can offer the timesaving, quality of results combined with the freedom to use your reagents and membranes of choice for immunodetection.”
Transferring samples from electrophoretic gel to the blotting membrane can take up to two hours with a traditional protocol, but Invitrogen has reduced transfer time to 7 – 10 minutes with their iBlot® Western Blotting System. The iBlot is a solid-phase (also called dry) blotting system that “eliminates the need for layering messy filter papers and sponges soaked with buffers, making the process much easier and clean-up free,” says Roumen Bogoev, marketing development manager for the protein analysis group at Invitrogen. “Not only is the iBlot easier to use and more reliable than wet and semi-dry blotting, using the iBlot can dramatically change your work flow and productivity. By using NuPAGE® Novex® gels, the iBlot system, and our ready to use WesternBreeze® kits, you can perform your Western analysis from gel to immunodetection within eight hours, rather than the 24+ hours required by conventional means.”
Multiplexing Westerns
Multiplexing, or the ability to make multiple measurements on the same sample simultaneously, is catching on in Western blotting—in part because it saves time and labor. “To avoid crossover reactions, researchers usually need to perform two Western blots (by either cutting the membrane into two halves or by stripping the membrane after the first blot and reprobing it) to detect the target protein and the internal control separately,” says Zhou. GenScript’s One-Step Western Multiplex Fluorescent Kit can detect four proteins on a blotting membrane simultaneously in about an hour.
LI-COR turns multiplexing and small sample sizes into a high-throughput approach to Western blotting with their MPX multiplexer blotting system. On a standard-size nitrocellulose blot, the MPX analyzes 24 sample channels, and it can detect twice as many targets using IRDye® infrared dyes. “Our MPX multiplexer blotting system works for both visible and infrared Western blotting applications allowing screening of multiple targets and multiple samples on the same blot in a high-throughput fashion,” says Jim Wiley, strategic marketing manager at LI-COR.
“One of the most exciting new developments lies in multiplexed, fluorescent Western blotting techniques,” says Priya Rangaraj, technical product manager at Thermo Fisher Scientific. In fact, Thermo’s new DyLight™ Dyes are incorporated into their DyLight™ Fluorescent Western Blotting Kits. Also new is Thermo’s Clean-Blot™ IP Detection Reagent. This reagent “is a novel secondary conjugate that improves detection of your target proteins because there is less interference from denatured IgG,” says Rangaraj. “The Clean-Blot Reagent is universal and works with antibodies from most species. It also readily adapts to your current protocol and buffers; simply switch your current secondary antibody conjugate to the Clean-Blot Reagent.”
Rockland Immunochemicals has developed antibodies especially for multiplexed fluorescent Western blotting. Their DyLight™-conjugated secondary antibodies “are prepared by coupling very bright and photo stable dyes to our outstanding line of highly cross-adsorbed secondary antibodies,” says Carl Ascoli, laboratory director at Rockland Immunochemicals. “This results in reagents that enable multiplex detection of several targets simultaneously in a Western blotting system.”
Antibodies galore
Rangaraj believes that “one of the biggest challenges facing scientists using Western blotting techniques today is finding primary antibodies that work reliably. There is a strong need for antibodies validated specifically for Western blotting techniques.” To meet this need, Thermo offers more than 100 siRNA-validated antibodies for Western blotting.
The importance of good antibodies in Western blotting should not be underestimated. “Western blotting is critically reliant on the use of antibodies for antigen binding, and propagation of signal for detection by a colorimetric of fluorometric tag,” says Ascoli. “The use of appropriate antibodies, both primary and secondary, with defined parameters for specificity and sensitivity is essential.” Rockland Immunochemicals validates their antibodies with both chemiluminescent and fluorescent detection, which Ascoli believes will become the norm. “Other antibody manufacturers will eventually follow our lead and provide users with antibody-based reagents accompanied with bench-side information on usage with actual testing results and conditions,” he says. “Our products are as thoroughly tested as reagent components used in a diagnostic setting.”
The Prestige Antibodies from Sigma-Aldrich are also validated “and are designed to have low cross-reactivity to other human proteins,” says Leigh Gaskill, market segment manager for antibodies at Sigma-Aldrich. Developed by the Human Proteome Resource along with Atlas Antibodies, the Prestige Antibodies are well suited for high-throughput applications. “A major challenge for researchers is the availability of validated antibodies specific to their target protein of interest that perform reliably,” says Gaskill. “Researchers are challenging antibody suppliers to develop more highly characterized, better-qualified antibodies.”
Quantification in the future
Many see a future for Western blotting in protein quantification. Wiley says that recent important developments in Western blotting involve “taking quantitation accuracy to new levels using the near-infrared as an imaging platform.” LI-COR’s Odyssey® Infrared Imaging System, for example, uses infrared fluorescence detection to give quantitative analysis and a wider dynamic range that chemiluminescence does not offer. In addition, says Rangaraj, Thermo’s DyLight 680 and 800 dyes, which detect in the infrared range, “are particularly attractive as they limit the autofluorescence from biological samples.” The combination of highly selective antibodies, faster blotting protocols, and quantitative imaging is allowing Western blotting to keep up with protein researchers, and to prove itself worth keeping.