Editorial Article
Wednesday June 09, 2010
by Caitlin Smith
Immunofluorescence has become such an important tool for biologists that one can now find it in almost any area, helping to illuminate the answers to questions big and small. Challenges for the technique include getting smaller (labeling distinct molecules) and more exact (labeling with validated antibodies). Here are several examples of the varied roads that immunofluorescence is traveling.
Validating antibodies for immunofluorescence
Cell Signaling Technology currently offers more than 700 primary antibodies validated for immunofluorescence (IF), according to Eric Scharf, director of marketing at Cell Signaling Technology. “Many of these are directly conjugated to Alexa Fluor® dyes, and all are validated in-house,” says Scharf. “In addition, Cell Signaling Technology now offers PathScan® Multi-Target HCA kits, which allow the user to assay eight signaling molecules grouped by pathway or biological process. Each antibody in this kit is optimized for automated IF applications in-house using high content platforms.”
Antibody validation at Cell Signaling Technology means that the antibody has been put through a defined, rigorous protocol that includes many applications. For example, the antibody will have been tested in Western blot, immunoprecipitation, immunohistochemistry, confocal immunofluorescence, flow cytometry, chromatin immunoprecipitation, and sandwich ELISA assays. The antibody undergoes a large battery of cellular tests as well. Antibody validation will also determine optimal conditions for the antibody to save to you time, such as protocol details, dilutions, and buffers.
Scharf says that Cell Signaling Technology is currently developing multiplex assays. This addition will likely help researchers take advantage of equipment such as automated imaging platforms. These have “revolutionized IF due to the speed and information capture,” says Scharf. “High quality antibodies from Cell Signaling Technology have allowed for refined detection of protein modification and mutations.”
The goal of Cell Signaling Technology, according to Scharf, is to provide customers with images clearly demonstrating correct protein activation and localization and outstanding performance; he says the biggest challenge today is the availability of rigorously validated antibodies for IF. “CST is addressing this challenge by understanding customers' needs and focusing on thorough antibody validation and new product lines for all IF applications,” says Scharf. “Moving forward, I see speed, resolution, and accuracy of the readouts improving drastically.”
More specific fluorescent tags
IBA’s Strep-tag® reagents enable researchers to study Strep-tagged proteins both in vivo and in interacting protein complexes. “The Strep-tag is a very commonly used tag in the field of recombinant protein production,” says Isabel Schuchardt, product manager for cloning, expression, and purification at IBA. “Due to its advantages based on its highly specific binding properties and the mild elution conditions, it allows the isolation of highly pure proteins and even protein complexes in one step. The newly offered, fluorescently conjugated monoclonal antibodies against the Strep-tag® represent an extension of the existing portfolio for recombinant protein production.”
Scientists are taking advantage of molecular tools, such as immunofluorescent tags, to discover the particular regions of individual molecules which interact together in binding complexes. “In my opinion, the biggest challenges in the field of immunofluorescence will emerge from science in order to understand and therefore visualize biological processes in more and more details, such as single molecule analysis,” says Schuchardt. In order to study smaller and smaller units of biological processes, though, detection technologies must continue to progress as well. “To fulfill this demand,” says Schuchardt, “dye companies will have to develop, on the one hand, increasingly sensitive and stable dyes to detect even weak signals; and on the other hand, optical systems are needed for the detection of these dyes.” For example, she notes new developments in FRET (fluorescence resonance energy transfer) technology for detecting protein-protein interactions, and in ChIP (chromatin immunoprecipitation) technology for detecting DNA-protein interactions. “The screening and verification of complex protein-protein and DNA-protein interactions within biological processes will be a major field of investigation in the future,” Schuchardt predicts.
Brighter, more tuneable fluorescent tags
Bruce Armitage, professor of chemistry at Carnegie Mellon University, works on developing fluorescent antibodies that are 10-fold brighter than current dye-labeled reagents. “Our approach involves the synthesis of DNA nanostructures that are functionalized with dozens of fluorescent dyes. We call these reagents ‘Fluorescent DNA Nanotags,’” he says. “We then attach several such nanostructures to a single antibody. By varying the dye that is attached to the DNA, we can tune the color of the nanotags.” Armitage also believes that the fluoromodule technology under development at the NIH Technology Center for Networks and Pathways (also at Carnegie Mellon University) is extremely promising. “This technology consists of fluorogenic dyes that are non-fluorescent in solution, and single chain antibody fragment proteins that bind to the dyes and cause them to light up,” says Armitage. “The protein component can be genetically encoded, analogous to GFP or other fluorescent proteins. However, since the dye is non-fluorescent when not bound to the protein, an excess of it can be present. The advantage over an inherently fluorescent protein like GFP is most evident in terms of photostability: once GFP undergoes photobleaching, it is destroyed. However, when a dye in one of our fluoromodules photobleaches, it can be exchanged with a fresh dye, allowing the fluorescence to be retained. We have developed a catalogue of different colors spanning the visible and near-infrared regions of the spectrum.”
Molecular Probes (of Life Technologies) is working with their Qdot® nanocrystals to develop a unique, tuneable set of fluorophores. Qdot® nanocrystals are tiny semiconductor particles that are actually clusters of atoms, containing from a few hundred to a few thousand atoms of a semiconductor material (cadmium mixed with selenium or tellurium). The particles are then coated with an additional semiconductor shell (zinc sulfide) to improve the optical properties of the material.
Qdot® nanocrystals have fluorescent properties that differ from typical dye molecules. The color of light that the Qdot® nanocrystals emit is determined mainly by their particle sizes—in other words, a range of sizes means a range of colors. However, their light emission peaks are narrow, symmetrical, and constant with different light sources, meaning their emissions can be sorted from one another easily (for example, in multispectral flow cytometry, with one to six colors). Further advantages of Qdot® nanocrystals compared to conventional dyes include their lack of photobleaching, and the inherent tuneability imbued by the range of particle sizes. Life Technologies offers new primary antibody conjugates to secondary detection reagents for flow cytometry, for combining Qdot® nanocrystals with existing organic fluorophores.