Editorial Article
Monday June 09, 2008
For about twenty years, there hasn't been any major evolution in the protocol for Western blotting. The same techniques and reagents have been handed down through the years. Recently, however, new products have begun to emerge to increase efficiency and convenience for the user. Current trends in Western blotting include fluorescent and chemiluminescent detection, accurate quantification, automation, self-cooling blotting systems, and widespread migration to digital, or CCD imaging systems. (CCD stands for charge-coupled device, an array of semiconductor gates that convert light to electrical charge.)
In fact, it is probably safe to assume that nearly all newer technologies for Western blotting will be built upon the foundation of CCD imaging instead of traditional film imaging. Film has a number of drawbacks. It can be time consuming, and requires a dark room and a developer. Film is expensive, and an ongoing cost. Also, it's somewhere between difficult and impossible to quantitate a protein band visualized on film—not really much better than "eyeballing" a coomassie-stained gel.
New Detection Chemistries
Chemiluminescent and fluorescent detection reagents can contribute substantially to the sensitivity, linearity, and dynamic range of Western blot detection. The Immune Star WesternC chemiluminescent kit is a new product from Bio-Rad that delivers the signal intensity and longevity that is optimized for CCD imaging of Western blots. When used with a CCD imaging system, it delivers femtomolar sensitivity. The Precision Plus Protein WesternC prestained chemilulminescent standards allow you to visualize your protein from the gel electrophoresis stage though the Western blot detection. The chemiluminescent signal is sharp, intense and will not cross react with your protein of interest.
Jeff Xu, global product manager for the Laboratory Separations Division at Bio-Rad, explains the company's approach to Western blotting products: "It all follows the trend within this market of offering products easier to use and with greater convenience for the customer, even though the overall protocol does not change." Another option on the market for chemiluminescent detection is Roche's Lumilight substrate. The system is based on luminol, a synthetic chemical that gives off light when oxidized. In the past, luminol-based chemiluminescent detection systems gave a very short-lived signal—about thirty minutes. Roche's newer reagents give a long-lasting signal, so there is no rush to detection, and linearity is good, even after dilution. Says Manfred Watzele, PhD, director in research and development for Roche Applied Science, "It has a very, very strong signal, allowing very sensitive detection. What also makes it suitable is the signal duration.... You can work relaxed and need not hurry to start the detection."
Meanwhile, Alpha Innotech is launching a new CCD-based imaging system for quantitative Western blot imaging and analysis that uses fluorescent detection reagents. The FluorChem Q combines the advantages of chemiluminescent imaging with the ability to image three different fluorescent dyes (such as Cy5, Cy3, and Cy2) in one quantitative Western imaging system. Using multiple fluorescent dyes for protein detection opens up possibilities such as detecting protein bands that overlap during electrophoresis and normally could not be distinguished on one blot if the researcher only used chemiluminescence. Says Lisa Valdin, marketing manager for Alpha Innotech: "Scientists these days aren't just asking is my protein there or not. They want to say, ‘how much of my protein is there, how much is present relative to a different protein, and what are the best practices for quantitating my results.’ Multicolor fluorescent Western blotting opens up new possibilities for the scientist doing multiprotein analysis."
Multicolor fluorescence can also increase the accuracy of quantitation on a blot. For example, if one fluorescent probe is used to detect a protein of interest, and a second fluorescent probe is used to normalize for sample loading control, you can quickly determine if the increase of your protein of interest in a particular lane is due to experimental factors, or loading inaccuracies. Whereas other fluorescent Western blotting systems depend on expensive laser detection systems, the Fluorchem Q delivers the same quality, but using a versatile CCD camera. It is designed as a standard multiuse imaging and not only captures images of traditional and fluorescent Westerns, but also DNA gels stained with ethidium bromide, protein gels stained with coomassie, and even colony plates—basically any imaging application in the lab.
Taming Transfer
Western blotting is a very straightforward method. When a problem arises, it often originates in the transfer step, rather than the incubation, development, and detection steps. Choosing good electrophoresis equipment, and the right kind of equipment for your application, can minimize the chance of something going wrong during transfer. There are three primary methods for carrying out a Western blot transfer: capillary (no electrical equipment); semi-dry, in which current is applied directly to the buffer-soaked blotter paper; and tank transfer where the gel is held in an open cassette and fully submerged.
Hoefer, Inc. offers electrophoretic transfer systems for most Western blotting applications. Tank models TE22 and
TE62 hold four cassettes, and have the capability to run at high voltages because they have the ability to cool through an external water bath. A tank model such as the TE22 or TE62 would be the best choice for transferring nucleic acids, because the buffers tend to be low ionic strength, and thus require a large amount of buffer.
If the quantity of buffer is an issue--containing hazardous chemicals, for example, or if it must be purchased at great expense, then a semi-dry transfer unit may be a good choice. The buffer volume may be as low as 50 mL. For a compromise between tank and semi-dry, Hoefer's "semi-wet" apparatus is a module that fits with the miniVE tank. It uses less buffer than a tank transfer, but more than a semi-dry transfer. "The unit is a hinged assembly that makes assembly, disassembly and cleaning easy. The feature of the modules is they fit within an existing apparatus reducing the number of instruments needed by a lab and allowing versatile performance from a single gel tank,"
says Nancy Laird, Senior Scientist at Hoefer, Inc. This option is currently not available for standard or large format gels, and cannot be run for extended periods due to the low buffer volume.
Automation for Westerns, finally
Western blotting is a labor intensive process containing several pipetting and wash steps, thus creating the opportunity for pipetting errors. The ProfiBlot™ family of instruments from Tecan is designed to improve the safety and reliability of Western blotting, as well as Southern blotting, by minimizing human exposure to potentially infectious samples. It is built to meet the IVD-directive 98/79/EC of the European Union. ProfiBlot™ 48 processes up to 48 Western blot assay strips per run, which are loaded into a 48-well tray. It automates tedious manual steps such as pipetting of reagents, incubation steps with integrated shaking, as well as wash steps. The user only has to pipette the samples. Additionally, the ProfiBlot™ 48 is compatible with an optional CCD camera from Tecan for easy documentation of the processed assay strips by storing the corresponding pictures, rather than by having to manually glue processed assay strips into a lab book.
Although for many years the technique of Western blotting has allowed us to take advantage of antibodies and immunogenicity to study the proteins and nucleic acids of the body, there is now greater interest than ever in studying the antibodies and the immune systems themselves. Additionally, the growth in diagnostic biomarkers has driven demand for clinical tools for identifying proteins and nucleic acids. Western blotting is an ideal tool for both of these applications, and with greater fidelity with regard to sensitivity and accuracy in quantification, not to mention convenience and ease-of-use, there are many excellent technologies to choose from.