Electrophoresis Apparatus, Gels Aid In Specialized Studies

FOR MULTIPLE RUNS: ISS’s Mini-6 Gel Device can run up to six gels simultaneously. In 1897, Russian physicist Ferdinand Ruess watched as clay colloidal particles moved within clay attached to electrodes, in what may have been the first application of what is today termed electrophoresis. In the 1930s, Arne Wilhelm Kaurin Tiselius developed methods to measure the amounts on an abnormal protein in the urine of multiple myeloma patients. That work earned him a Nobel Prize in 1948, and the detection methods vaulted electrophoresis to a critical position in biological research. Electrophoresis has come a long way since scientists first took notice of the effects electric fields have on matter. "Virtually every bio laboratory uses an electrophoretic method," says Johann Bauer, a cell biologist at the Max Planck Institute for Biochemistry at Martinsried, Germany, and author of Cell Electrophoresis (Boca Raton, Fla., CRC Press, 1994). Electrophoresis can handle separation of just about any charged particles, from charged small molecules to biological polymers like RNA, DNA, and proteins, and even whole cells. Prior to the 1950s and the explosion of genetic research, electrophoresis was commonly applied to separation of lipoproteins, carbohydrates, polysaccharides, hormones, vitamins, and the occasional protein or nucleic acid mixture. Today it is used primarily to separate nucleic acids and proteins. The technique owes its utility to the tendency of charged particles-once subjected to an electric field-to migrate between two oppositely charged poles. The matrix that the molecules flow through-referred to as a gel-is the real player, though. The pores of the gel restrict larger molecules, slowing them down. Take DNA or RNA separation: Agarose (isolated from kelp) is the gel of choice for quick separations, readily separating large fragments (ranging from 125 to 2,500 base pairs) from smaller ones. But because agarose is coarse, it can't distinguish sequences with slight variations. That's where polyacrylamide comes in. Its finer structure can distinguish nucleotide stretches varying by only one base pair, making polyacrylamide the gel of choice for DNA sequencing. But the cross-linking of bis-acrylamide is disrupted by oxygen, so acrylamide gels are cast between two vertical glass plates to seal it from air. The gels are also run vertically. Integrated Separation Systems Inc. (ISS) of Natick, Mass., offers its Mini 6-Gel Device for the researcher who has a need for multiple acrylamide runs. It's the only vertical gel device that can run up to six gels simultaneously, according to Margaret Mary DeRoo, marketing manager at ISS. "Unlike conventional systems with an upper cathode and lower anode buffer chamber, full contact of the gel plates with both buffer chambers allows efficient temperature control and 'smile'-free gels," says DeRoo. Such an aberrant gel "smiles" back at the researcher-with center bands lower than the peripheral ones-usually to the detriment of separation. These Cheshire cat aberrations result from differing temperatures-and hence different flow rates-along the gel. FOR ISOLATING DNA: MacConnell Research Corp.’s Mini-Prep 24 reportedly produces sequencable-grade plasmid DNA in one hour. The Human Genome Project is creating a huge volume of DNA sequencing research that cries out for automation. MacConnell Research Corp. of San Diego is looking to the DNA isolation sector for users of its Mini-Prep 24, designed to isolate DNA from bacteria cultures (commonly used in the cloning and amplification of human genes). "A mini-prep from E. coli [bacteria] is the beginning stage of almost any transcription exercise," explains Bill MacConnell, the company CEO. This system produces sequencable grade plasmid DNA in one hour, according to company literature. The instrument lyses the bacteria in the culture, loads soluble molecules into pre-cast agarose gels, and-through electrophoresis-separates DNA from the other soluble bits from a lysed bacteria. The DNA, once collected, is ready for sequencing. "DNA isolation from bacterial cultures represents the largest market segment of DNA purification at present," reports MacConnell. "The task of DNA plasmid purification consumes countless technician hours, [since] nearly all recombinant DNA applications require purification of plasmid DNA." Selected Vendors of Electrophoresis Equipment and Supplies: Beckman Instruments Inc. Boehringer Mannheim Corp. C.B.S. Scientific Co. Inc. FMC Corporation Helena Laboratories Integrated Separation Systems Inc. MacConnell Research Corp. Owl Scientific Inc. Sooner Scientific Inc. Stratagene Corporation Protein separation is the other major use of electrophoresis. Like DNA, proteins are often separated by size alone. But di-sulfide bonds often interconnect the chains of a protein, and since that can interfere with separation, protein electrophoresis is usually done in the presence of reducing agents like sodium dodecyl sulfate (SDS). Once the proteins are separated, the researcher often applies coomassie blue stain to the proteins within the gels. Alternatively, the proteins can be "blotted" onto a separate membrane. Once the proteins are immobilized, the researcher can apply a protein-specific antibody-itself linked to an enzyme. That enzyme, in turn, converts an applied solution to a fluorescor or a blue precipitate (depending on the enzyme system used), either of which confirms the presence of the protein. But sometimes size separation alone isn't enough. In those cases, isoelectric focusing is typically the first step. Placed in gelatin-like agarose or polyacrylamide, a protein will migrate within a gradient of increasing pH until it reaches a spot where the net charge of all its side-chains is 0 (the isoelectric point). A molecule's unique isoelectric point is determined by the number and nature (+ or -) of the charges, the size of the molecule, and its overall shape (since exposed charges are more responsive to the field). Because proteins incorporate 20 commonly found varieties of amino acids-and five of these may bear a positive, negative, or neutral charge, depending on the pH-there is enough variation between proteins for them to behave differently in isoelectric focusing. DNA and RNA are not amenable to separation by isoelectric focusing because the base pairs all have very similar charges. There may be more than one protein in a sample with very similar isoelectric points, so proteins are often subjected to further separation based on size in a process called 2-D electrophoresis. Here the vertical line of the run-with its accompanying bands where protein components have settled-is rotated so that it is horizontal, and then is placed in a new gel to serve as the baseline. This time the separation depends on the ratio of the protein's size to its overall charge. The simplicity of SDS 2-D electrophoresis comes at a price: The researcher must compare the lanes generated with markers to get a rough estimate of the protein's size. That frustrates researchers like Carl Merril, chief of the Laboratory of Biochemical Genetics at the National Institute of Medical Health and editor-in-chief of the journal Applied and Theoretical Electrophoresis. Merril and others have embarked on research to link mass spectroscopy-a technique that generates the precise molecular weight and telltale decomposition patterns from ionized molecules-to electrophoresis. Many researchers envision using lasers to ionize proteins on the surface of a completed gel, followed by mass spectral analysis to determine the protein's size. "If the mass spec can be used in this manner, you could get very accurate masses, and from the intensity of the signal you should be able to get quantitation, too," he explains. Since proteins often decompose one amino acid residue at a time, the mass spectral analysis also has the potential to provide the mass and sequence of the protein in one fell swoop. This quest has been fueled in part by what Merril terms the "proteome project." which will add to the information gained from the Human Genome Project. The Human Genome Project will eventually provide a map of the proteins generated from the myriad genes of the human genome, but it "would be very hard at this point to predict when those proteins are expressed, or the quantity expressed, because there are external forces involved," Merril says. A researcher interested in the pituitary gland might remove the gland from inbred mice at different stages of their life cycles and then analyze the identity and quantity of the proteins present. That could give insight into how protein patterns change during development. Just as the Human Genome Project is creating a demand for automation of gel electrophoresis to meet the huge demand for sequencing, Merril believes proteome research could demand big steps forward in mass spectral analysis. A rapid and accurate method to read the molecular mass from an electrophoresis gel would make it much simpler to identify proteins present in a complex system, and that would be a boon to the project. In the meantime, companies are focusing on improving gels and techniques for the most common uses of electrophoresis. According to Bauer, electrophoresis product development is occurring at "rather high speed, aiming to enhance resolution, speed, and throughput of the various electrophoretic methods." CHEMISTRY KITS: Each Beckman eCAP gel is optimized for different kinds of samples Labs, of course, come in all shapes and sizes. Teaching and research labs-especially in academia, where money is tight-are happy with manual gels prepared and run by students and researchers. It's a slow and arduous process-good for building character in students and researchers, perhaps-but the gels produced may vary in quality. Diagnostics and quality-control applications require high throughput, and they demand consistency of results. That's where companies like Beckman Instruments Inc. of Fullerton, Calif., come in. Beckman's P/ACE 5000 series, combined with the eCAP chemistry kits the company sells separately, offer technical simplicity and flexibility, according to company literature. QUICK ANSWERS: Beckman Instruments’ PACE 5000 system offers speedy acquisition, according to the company. In place of a flat gel, the instrument uses narrow capillary tubes (of 20 micrometers to 200 micrometers internal diameter), which can be filled with various eCAP gels, each optimized for different kinds of samples. These plug-ins include the capillary, standards, buffers, and methods for separating: chiral (or "handed") small molecules, amine small molecules, DNA, and proteins. The tubular structure of capillary electrophoresis allows easy detection of outflowing analytes: Depending on his or her needs, a researcher can choose from four detector options. Beckman thinks P/ACE could be a boon to harried quality-control scientists. Quality-control managers "will ask for data to be presented at a 3:00 conference, but they usually tell you about it at 11:30," quips Tom Pritchett, principal scientist at Beckman. That may be an exaggeration, but quality control often requires quick decisions and that, in turn, demands fast data acquisition. Traditional gels may require half a day by the time the gels are prepared, loaded, run, and finally detected. The P/ACE 5000 promises answers in as little as 30 minutes. IN THE CLINIC: Helena Laboratories' the Cardio REP device is an example of a clinical use of automated electrophoresis. Automated electrophoresis is also making its way into clinical use. Helena Laboratories Inc. of Beaumont, Texas, offers the Cardio REP device, which can help diagnose the extent of damage in heart muscle in the critical early hours following a heart attack. The system measures the levels of two isoforms of heart-tissue-associated creatine kinase. The tissue form, called MB2, is released from heart cells as they necrose. Once in the bloodstream, a protease alters the enzyme to isoform MB1. The Cardio REP measures the ratio of the two isoforms. MB2 makes its way into the bloodstream naturally, and normal ratios of MB2-to-MB1 fall in the 1-to-1.5 range. A ratio greater than 1-to- 1.7 suggests a recent outpour of MB2 from damaged heart tissue, and that suggests a heart attack. Physicians can get a complete profile in 24 minutes, according to company literature. Helena also offers a similar system for detecting cholesterol levels. Other less costly convenience products may appeal to a broader audience. Take Castaway pre-cast sequencing gels, sold by Stratagene Corp. of La Jolla, Calif., for instance. These acrylamide gels-large enough for DNA sequencing applications-come pre-made, ready for sample loading. That alone can save a lot of time: Mixing solutions, cleaning plates, de-gasing acrylamide, pouring the gel, and waiting for it to polymerize can take up to two hours. "We are the first to overcome the issues of stability and consistency for the larger-format gels used for sequencing," claims Douglas Drake, product manager at Stratagene. "No one else manufactures precast sequencing gels. They are stable for two months at 4°C." And bis-acrylamide presents problems of its own. "Unpolymerized acrylamide is a neurotoxin. [Castaway gels] help prevent exposure to it," says Drake. READY-TO-POUR: Sooner Scientific Inc.’s SeQuate acrylamide gel mixture For those interested in greater simplicity of gel preparation, but still willing to do some of the work themselves, Owl Scientific Inc. of Woburn, Mass., offers its Burst-Pak sequencing gels. These packets contain all the materials required to polymerize the acrylamide. After breaking the internal seals, the researcher pours the gel into his or her plate assembly. The gel reportedly will polymerize in about 15 minutes. Owl also makes pre-cast acrylamide gels that are suitable for protein or DNA electrophoresis. Sooner Scientific Inc. of Garvin, Okla., also offers a ready-to-pour acrylamide gel mixture called SeaQuate. SIMPLIFIES PREPARATION: Owl Scientific’s Burst-Pak gels contain all the materials required to polymerize acrylamide. Stockpiling equipment for later use can ease the burden of a long day in the lab, and to that end, C.B.S. Scientific Co. Inc. of Del Mar, Calif., offers Mini Horizontal Gel Casters. With them, a researcher can pre-cast up to six mini agarose gels at a time. Automation and ease of use for traditional applications continue to be a main focus of many companies, while others have focused on introducing new types of gels better suited to specific applications. Boehringer Mannheim Corp. of Indianapolis offers agarose MP (short for "multi-purpose") and agarose LM-MP (short for "low-melting, multi-purpose"). Both are tailored for use with pulsed-field gel electrophoresis (PFGE). This technique can separate large DNA fragments greater than 10 Mb in size. Large DNA fragments are normally run in the coarser agarose gel, but it is unable to distinguish small size differences in the DNA. But large DNA fragments need time to reorient themselves after an electric pulse, before they continue migrating along a plate. PFGE inverts two electric fields in a series of pulses, and the highly size-dependent reorientation time produces good separation among similar-sized DNAs. "PFGE is crucial to the Human Genome Project [and] has become one of the main methods to characterize large DNA," notes Sandy Miller, a molecular biologist at Boehringer Mannheim. "Being able to run the large fragments directly [instead of digesting them first] is definitely a time-saver." ProSieve 50-a proprietary acrylamide gel formation offered by FMC Corp. of Rockland, Maine-yields protein separation comparable to separations on gradient concentration acrylamide. Changes in gel concentration cause changes in pore size, and gradients accentuate small differences in size. But laying down gradient concentrations is a time-consuming process, making ProSieve 50 an attractive alternative for some applications. Point mutations frequently confound conventional acrylamide, since the mutant is often the same size as the normal DNA. FMC's MDE (Mutation Detection Enhancement) gels promise greater sensitivity to mutations, relying upon secondary structure of DNA to pick them out. The interactions involving small molecules and solid supports are not well understood, and the MDE gels are no exception. "What [the interactions] are is something of a mystery," says Mark M. Gardner, senior research scientist at FMC. "There's no simple, direct way of going from molecular conformation to relative electrophoretic mobility." Such a predictor may lead to an explosion in new designer gels, but until then, researchers and companies will continue to refine the tried-and-true methods. The Human Genome Project and its companion proteome project will provide plenty of demand for better and more efficient electrophoresis apparatus. James Kling is a science writer based in Bellingham, Wash.

Electrophoresis Apparatus, Gels Aid In Specialized Studies

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