Automation, new detection methods give the old favorite Western blotting a new look.
Calling it a ”Western blot” was a joke, actually. It was inspired by the “Northern blot”, which itself is a play on “Southern blot,” invented by Edwin Southern in 1975—and sensibly named after him. So it’s sort of like the joke made by someone at a cocktail party who doesn’t know that everyone else is done laughing and has moved on.
That someone was W. Neal Burnette (although the invention of the technique is widely credited to George Stark). Burnette probably wishes he had played it straight and named it the “Burnette blot”, because it’s the Western blot that continues to be used and loved, and referenced by scientists the world over, whereas the Southern and Northern have become museum curiosities. Younger scientists generally have no experience with Southern and Northern blots (since there are faster and more powerful ways to detect DNA), and need to have it explained to them why their immunoblot is named after an omelet. Why do we cling to the Western, in the age of rapid proteomics, and should we?
The rise and fall of Western … blotting?
Mathias Mann of the Max Planck Institute of Biochemistry (Germany) has called for the Western to join its compass-themed brethren in mothballs, in “Can Proteomics Retire the Western Blot?” (J. Proteome Research. 2008 7(8): 3065.) Mann writes, “Biologists currently are stuck in a time warp—they basically use the same tools for protein detection as they have for the past 20 years.” The column makes a persuasive argument that the Western blot method has defined and limited the work of researchers using the technique, and that methods such as mass spectrometry would result in a “paradigm shift” in biology.
Certainly, improvements in mass spectrometry in combination with powerful bioinformatics applications make it possible to run a profile of a complex sample and identify every protein in it. The most obvious impediments to the widespread adoption of mass spectrometry as a replacement for the Western are cost and convenience. In time, mass spec may become even more convenient and economical than the Western. However, that time is not now.
More significantly, mass spectrometry has a hard limitation in the dynamic range of protein concentrations it can detect in a single sample—typically only about 3 to 4 logs. However, the relative abundance range of biologically-significant proteins in nature is much larger than that—widely estimated at around 12 logs in a sample such as human blood. This means that the Western blot isn’t going anywhere right now. We need it, if only to confirm the results of our big mass spec experiments.
So, granted, some users will only give up the Western blot when it is pried from their cold, dead hands. But that doesn’t mean they have to use the same Western that their Mom used in her research lab back in the 70s. Improvements in detection chemistries, antibodies, CCD cameras, software analysis, and robotics can bring the modern user’s Western blot into the 21st century, as well as address some of those same deficiencies that have experts calling for it to be banished.
Quantitative after all
One of the major disadvantages of Western blotting is—all together, now—it’s not quantitative. The immunoblot can be exquisitely sensitive, but one will never know how sensitive until they’ve quantified their sample separately (often using methods that give only a rough estimate and consume valuable sample). The advent of CCD imaging has changed the landscape dramatically when it comes to quantification of samples on blots and gels. For example, Bio-Rad (Hercules, Calif.) has recently released its Western C chemiluminescent detection kit for Western blotting. The use of the chemiluminescent reagent plus a CCD imager gives the blot femtomolar sensitivity for detection, plus reliable quantification from a highly linear standard curve. Digital images are also cheaper than film. Says Jeff Xu, global product manager for the Laboratory Separations Division at Bio-Rad, “The main advantage of the CCD imaging computer is that it gives you a lot of leeway to optimize your image without costing a lot of money.”
Many vendors are offering Western blot detection kits optimized for convenience, sensitivity, and accurate quantification. Roche’s Lumi-Light system uses luminol, a synthetic chemical that gives off light when oxidized. Roche Applied Science (Indianapolis, Ind.) also optimized its reagents in order to overcome the challenge of the typically very short-lived signal from the luminol. Taking the concept of convenient, quantitative detection a step further are systems such as the FluorchemQ from Alpha Innotech (San Leandro, Calif.), which integrates the CCD imaging system with a multi-channel detection system that can handle three dyes at a time.
Says Lisa Valdin, marketing manager for Alpha Innotech, “In the old days of chemiluminescence, the standard was you’d use HRP, or AP, you’d … detect it with film … that was technology from 10 years ago. CCD technology entered the realm within the last 10 years. What CCD offered was you could still do chemiluminescence, but you eliminated the need for film.” Film did not have a wide dynamic range. And if the protein concentrations exceeded the linear range for the film, the bands might look equally intense, making any estimation of protein concentration inaccurate. Says Valdin, “What a CCD imager allows you to do is have a wide linear dynamic range. What dynamic range allows you to do is see faint bands at the same time as a bright band.”
The multi-channel detection capability of an instrument like the FluorchemQ opens up some new options for separating proteins. Using multiple dyes lets researchers distinguish between phosphorylated and non-phosphorylated forms of the same protein, for example. Dmitry Bochkariov, principal scientist for Advansta, Inc., Menlo Park, Calif., is working on projects with the National Cancer Institute to develop cancer-specific biomarkers. Recently his laboratory tested FluorchemQ for the simultaneous detection of phosphorylated and non-phosphorylated proteins, using two dyes and an antibody specific for the phosphorylated state. Regarding the outcome, Bochkariov says, “It’s doable, if you know the appropriate conditions on the specific protein you’re looking for.”
Blotting on autopilot
Improving the scientific performance of the Western blot is only one part of the equation. Western blotting can be time consuming and labor intensive, when one considers the process from beginning to end. Automated systems are turning up all over the biological laboratory, helping with tasks from sample prep to high throughput screening. Fortunately, there are some automated systems out there for Western blotting.
Blue Sky Biotech (Worcester, Mass.), uses a wide variety of proteomics technologies in their contract work. Their primary need in protein detection is to confirm the identity of a protein, rather than whole proteome scanning or biomarker discovery. For this application, they maximize their throughput using Western blotting machines that automatically carry out washes, primary and secondary incubations, and other time-consuming liquid handling tasks. Because they are able to automate the Western blot procedure, it can be done overnight, making more efficient use of laboratory time. Says Scott Gridley, PhD, director of protein sciences for Blue Sky Biotech, “When we come in, the membrane is ready for detection.”
There is no denying the fact that nearly everyone would rather squirt their sample into a mass spectrometer (or another prep-free proteomics platform) and quickly and accurately identify every protein in the sample. To some extent, this dream has been realized for nucleic acid applications, but on the proteomics side, significant barriers remain. Until a comprehensive, cheap, and fast proteomics platform comes along to replace it, we are stuck with the Western blot. We might as well get used to it.
About the Author
Catherine Shaffer is a freelance science writer specializing in biotechnology and related disciplines with a background in laboratory research in the pharmaceutical industry.
This article was published in Drug Discovery & Development magazine: Vol. 11, No. 10, October, 2008, pp. 34-37.
Filed Under: Genomics/Proteomics