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Gel Documentation and Analysis Systems Gel Documentation and Analysis Systems (continued) Alpha Innotech's FluorChem His name was Herman. The kindly, silver-haired Estonian immigrant ran the department photography shop. Anybody needing to capture an image of a Coomassie or silver-stained protein gel let Herman work his magic with light box, camera, and developing solutions. He started by asking the researcher to carefully position the gel on his light box. Next, he installed the proper filter and made the necessary f-stop settings on the camera. He then crawled under the camera's black hood to peer thoughtfully at the arrangement. When he was finally satisfied, he swiftly lifted the lens cap away for a second or two, then deftly replaced it with a flick of the hand. In a fluid movement, Herman pulled the film cassette from the camera and vanished into the darkroom. The researcher was left to wait with some old magazines while muffled sounds and grunts emanated from the dark. Nowadays, the life scientist has access to many gel documentation and analysis systems. Built around scanning densitometers or state-of-the-art charge-coupled device (CCD) cameras, these devices have brought gel documentation and analysis a long way since Herman's time 15 years ago. Certainly there is no shortage of system choices. The number of available systems and the market competition for imaging dollars speaks loudly about the utility of gel documentation and imaging systems. Here, LabConsumer takes a closer look at the imaging and analysis of protein gels. Stratagene's Eagle Eye Jr. System Anatomy The typical CCD camera-based imaging system has several components. The heart of the system, the CCD camera, is usually mounted on top of or within a benchtop darkroom or cabinet. The camera itself can be standard, integrating, or cooled, often with a manual or motorized zoom lens. (LabConsumer recently described the inner workings of CCD cameras and CCD-based imaging systems.1) The sample to be imaged is placed on a UV or white light box within the benchtop darkroom. For lower-cost, basic imaging systems, the image is viewed directly on a black and white monitor before printing on a thermal printer. More advanced systems employ features such as frame grabbers and sophisticated software packages for use with IBM-compatible or Macintosh computers. These systems allow image analysis, such as molecular weight calculation and quantitation, as well as image archiving for database and proteomics applications. Researchers can buy systems fully configured or customized with various options and components. The systems built around scanners usually consist of the densitometer and its controlling computer. The computer also runs the software for image analysis. Available densitometers have reflectance and/or transmission capabilities designed to scan both transparent and opaque samples. These include wet and dry gels, film, and membrane blots. Of course, the imaging systems vary in their complexities and capabilities. The low-cost, entry-level systems are typically billed as economical replacements for instant film. Considering that a case of Polaroid Type 667 film can cost more than $700, such systems as the FOTO/Analyst from FOTODYNE, the GeneCam Flexi from Syngene, and the EDAS 120 from Eastman Kodak would soon pay for themselves in film cost savings. UVP's Fluor Chemi System Conversely, researchers can spend $40,000 and more for sophisticated, multifunctional, and specialty imaging systems. Fuji Medical Systems' LAS-1000plus, for example, features an electronically cooled 1.3 million-pixel CCD camera with one of the industry's highest quantitation performances, crossing four orders of magnitude in its linear range. The LAS-1000plus captures chemiluminescent, fluorescent, luminescent, and colorimetric images and performs densitometric analyses. The majority of the gel documentation and analysis systems are priced between $4,000 and $30,000. Offering simple image acquisition and printing to image analysis and archiving, these systems cross the spectrum of imaging needs. Under Analysis After Herman emerged from the darkroom, he carefully examined the freshly developed negatives. If they met his exacting requirements, the photographic prints of the gel usually appeared in the researcher's mailbox a few days later. A good set of eyes and a ruler helped in the analysis, and Margaret, the department illustrator, applied the necessary annotation, such as molecular weights and lane numbers. That was largely the extent of gel documentation and analysis not so long ago. Now powerful software packages enable the life scientist to improve and clarify gel images in many ways. To begin with, the image-capture process itself is subject to various alternatives, such as dynamic integration, on-chip integration, and frame accumulation. After capture, images can be optimized to equalize brightness and contrast, sharpened to define unclear edges of bands, background subtracted, and subjected to "star field correction," a process that removes unwanted "hot pixels" and noise. Annotations in the form of text, lines, shapes, and colors are readily added to any image. Quantitative image analysis follows. Highly capable software packages for the analysis of 1-D electrophoretic images, such as ONE-Dscan from Scanalytics, employ automatic lane-finding routines that use pattern recognition and artificial intelligence techniques to detect band features and lane patterns. Bands are then quantified in terms of integrated density, molecular weight, size, band migration, retardation factor (Rf), and concentration. Other packages, including AlphaMatch(tm) 2D from Alpha Innotech Corp., tackle the analysis of 2-D gels. To eliminate the tedium of manually comparing gels, the software programs use various algorithms and landmarking techniques to match spots between separate gels. Database functions in turn offer the comprehensive interrogation of 2-D gel data sets so that data fields such as gel conditions, sample identification, and mutation can be searched and correlated with gel-derived data such as molecular weights and isoelectric points. The inherent variability of electrophoresis complicates comparisons between gels, however. Consequently, UVP Inc. of Upland, Calif., puts considerable effort into the development of editing and correction features in gel analysis software. Doug Kelley, director of sales for UVP, explains, "Our goal is to determine Rf markers so that we can get gel-to-gel correlations." UVP's software uses specific bands to calculate Rf marker bands. Such bands, when common to several gels, can be reference points to compare gels that have been run to different lengths. "By having software that allows editing and corrections, users have much more confidence that results obtained through databasing and querying are accurate. Researchers are able to base some assumptions on what they have found without doing a lot more experimentation." The ability to compare many gels is important in proteomics, so Ann Arbor, Mich.-based Genomic Solutions sells the Investigator(tm) Proteomic Analyzer Camera System. Equipped with a high-resolution CCD camera and a benchtop darkroom designed to acquire images of gels stained with fluorescent and colorimetric dyes, the Investigator Camera System uses powerful Investigator HT Proteomic Analyzer software to analyze 2-D gels. Designed for high-throughput applications, the HT Proteomic Analyzer software overcomes the research bottleneck of 2-D gel analysis by enabling the processing of up to 50 gels per day. What's more, the system tackles gels with over 10,000 spots and provides information to the Flexys(tm) Proteomics, a device that automatically excises proteins from gels. According to Mary Lopez, vice president of proteomics research and development at Genomic Solutions, the company focuses on products for genomics and proteomics research applications. Indeed, the Proteomic Analyzer Camera System is part of the company's "full circle" of integrated proteomics research tools. Known collectively as the Investigator Proteomic Solution, these integrated tools handle everything from 2-D gel running to automated gel staining, spot picking, and sample preparation for mass spectometry. Scanalytics' imaging capabilites Stain Game Acquiring images of gels stained with Coomassie Blue or silver is standard duty for gel documentation systems. Many different Coomassie Blue and silver-based staining products for protein gels are available,2 and none of the companies contacted for this profile reported any particular difficulties with their use in standard imaging applications. The preponderance of imaging systems that can acquire images of fluorescent samples, on the other hand, was quite striking. Most of the CCD-based imaging systems can excite and capture images of protein gels stained with fluorescent dyes such as Molecular Probes' SYPRO(r) Orange and SYPRO Red. Frequently, the scanner-based systems that detect fluorescent samples have multifluorescent color capabilities as well. The Typhoon 8600 from Molecular Dynamics, for instance, detects four colors simultaneously. The FluorImager 595 and Storm 860, also from Molecular Dynamics, detect two colors simultaneously. The FMBIO II from Hitachi Genetic Systems detects up to 23 different fluorescent stains and labels. Use of fluorescent dyes has grown considerably in recent years. Peter McNamara, staff scientist at Molecular Dynamics, explains. "In the last five years, we have seen an explosion in options for fluorescent gel stains as well as other fluorescent labels and tags. The technology has gotten better in the form of kits and procedures, and these labels and tags have become useful tools that are much more sensitive than the traditional methods. For protein gels, the use of fluorescent stains is an extension of the use of ethidium bromide as a standard stain for agarose gels. The staining protocols for fluorescent stains are very similar to Coomassie Blue, something most labs are already very familiar with." SYPRO Red and SYPRO Orange dyes, products of Eugene, Ore.-based Molecular Probes, were frequently mentioned as gel stains readily imaged by fluorescent gel documentation systems. These stains have sensitivity levels comparable to colloidal Coomassie Blue and some of the rapid silver-staining methods. One of Molecular Probes' newer stains, SYPRO Ruby dye, matches the sensitivity of the best silver stains and is being applied in proteomics. "Fluorescence is becoming particularly important to high-throughput, automated proteomics," explains Wayne Patton, group leader of bioanalytical assay for Molecular Probes. "Fluorescent dyes that do not covalently modify proteins are finding application in the downstream microchemistry that is necessary in proteomics, especially mass spectrometry." Patton explains that the linear dynamic range of SYPRO Ruby dye extends over three to four orders of magnitude, compared to essentially one order of magnitude for Coomassie Blue and silver stains. As a result, SYPRO Ruby dye permits the accurate quantification of subnanogram to multiple microgram amounts of proteins on a single gel. With excitation peaks at 300 and 470 nm and emission maximum at 610 nm, SYPRO Ruby dye is compatible with a wide range of imaging instrumentation and many of the new visible blue light imaging boxes.3 Recently, Molecular Probes introduced SYPRO Tangerine dye, a nonfixative stain similar to SYPRO Orange and Red dyes in sensitivity. Designed to stain gels in a saline solution instead of acetic acid or organic solvents, SYPRO Tangerine dye enables the visualization of proteins without interfering with western blotting, zymography, electroelution, or mass spectrometry. Improving Your Image So what can a researcher do to maximize the return from gel imaging and analysis? Many factors contribute. Among these are gel type and concentration, sample preparation, band size, dye concentration, and staining procedures. A good starting point is to always use good raw materials. Run gels with well-separated lanes and strive for good band resolution. Run standards of known quantity or a dilution series, if possible, to produce a standard curve. Bear in mind the linear range of the system. Faint or saturated bands often yield inaccurate results. Additionally, use consistent staining and destaining protocols and avoid excessive staining. When comparing gels, ensure that all variables are as similar as possible. Fluorescent gel stains present their difficulties, too. "The use of fluorescent stains is an area that many of our customers need help with," says McNamara. "It is not necessarily a seamless transition to go from traditional methods, such as isotopic detection, to a fluorescent scheme. Background is one problem area because the staining and destaining procedures for fluorescent gel stains can require some optimization for best use." McNamara further explains that particulate specks sometimes appear on fluorescently stained gels, probably caused by small, insoluble bits of the stain. Molecular Dynamics' sensitive gel scanners detect these specks more readily than UV transilluminator and camera systems. "Even though it is still very easy to get good results with good sensitivity, the artifacts caused by the specks will occasionally complicate quantitation," he says. A few precautions can improve the imaging of fluorescent gels. First, powder from gloves is fluorescent, so handle gels with powder-free gloves or gloves rinsed with distilled water. Also, thoroughly clean all containers and glass plates, ensuring they are free of grease and fingerprints. Protect fluorescent stains from exposure to light to prevent photobleaching of the dyes, ensure that dye stock solutions are completely dissolved, and use fresh staining solutions. Finally, when scanning, place gels on low-fluorescence glass plates or sample holders. Avoid plastic wrap unless it has been tested for low background fluorescence. The accompanying table lists many of the available systems for protein gel imaging. With all of these options, it is exceedingly clear that no protein gel is safe from capture these days. Michael D. Brush can be contacted at mdbrush@compuserve.com. References 1. G. Meisenholder, "Reality bytes: digital imaging systems take the guesswork out of data analysis," The Scientist, 13[17]:20-24, Aug. 30, 1999. 2. M. Brush, "Dye hard: protein gel staining products," The Scientist, 12[10]:16-18, 22-23, May 11, 1998. 3. K. Berggren, "A luminescent ruthenium complex for ultrasensitive detection of proteins immobilized on membrane supports," Analytical Biochemistry, 276:129-43, Dec. 15, 1999.

Strike Up the Band

Feb 20, 2000

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