Molecular Size Detector Increases Protein Purification

Most life scientists, at some point in their careers, are faced with the problem of purifying a specific protein from a biological solution. For example, they may need to purify an antibody from a serum, or a single E. coli protein from an extract. The separation of a single protein from a mixture of thousands is a considerable task - it takes a long time to collect enough raw materials to make purification worthwhile. Purification is necessary, however, to provide valuable information about the exact chemical and physical nature of the protein under investigation, such as its molecular weight, structure, and biological activity. Throughout the years, researchers have developed a number of different techniques for purifying large quantities of a specific protein from a protein mixture for further study. One of the more popular methods of preparative protein purification is liquid chromatography. Most liquid chromatography separations are performed through the use of automated systems that allow for rapid and efficient protein purification. A heterogeneous mixture of proteins in solution, such as a cell extract, passes through a column that separates proteins on the basis of their specific molecular properties, including size, shape, and charge. The eluate from the column flows first through an ultraviolet (UV) spectrophotometer, which records the presence and concentration of each protein as a corresponding peak on a chart recorder. The liquid is then collected in small fractions. Each column fraction must be further analyzed (usually by gel electrophoresis) for purity and molecular weight before the purification can be completed. Using gel electrophoresis, however, can be time-consuming and costly. It generally takes between one and two days to perform the procedure, resulting in considerable labor costs in addition to the price of the apparatus. A recent advancement in the science of protein chromatography provides researchers with an alternative to lengthy protein assays. Oros Instruments has developed the Model 801 Molecular Size Detector, an on-line instrument that can measure the molecular weight and purity of proteins immediately after their elution from a liquid chromatography system. Scientists can connect this free-standing, benchtop instrument to any liquid chromatography system, including HPLC (high-pressure liquid chromatography) and FPLC (fast protein liquid chromatography). At present, Oros' unit is the only device of its kind on the market. The molecular size detector is based on the principles of photon correlation spectroscopy, or dynamic light scattering. Molecules in solution undergo Brownian motion, a constant random movement. The rate at which the molecules move is directly related to their size; that is, small molecules move more rapidly than large ones. In the molecular size detector, light from a miniature laser passes through a flow cell containing the sample. Brownian motion of the molecules in solution scatters the photons of laser light as they pass through the sample. The movement of the molecules relative to each other produces constructive and destructive interference, which causes a fluctuation in the intensity of the scattered light. A sensitive photodetector detects this fluctuation and measures it over time. A digital signal processor within the instrument receives the light intensity information and analyzes it using a complicated mathematical manipulation. This program provides the hydrodynamic radius, or average molecular size, of the protein. Once the hydrodynamic radius is known, the instrument's computer calculates the molecular weight. In addition, the instrument provides an assessment of purity and concentration by measuring the size uniformity of the molecules in the sample. The Model 801 Molecular Size Detector also detects the presence of protein aggregates and degradation products. A comparison of literature values of protein molecular weight and those determined by the instrument shows good correlation for proteins with a molecular weight between 12,000 and 50 million. The Model 801 Molecular Size Detector, which costs $17,500, is compact and fully automatic, making it compatible with many different liquid chromatography systems. Researchers can also use it as a stand-alone instrument for manual molecular size and weight determinations. When the detector is used in tandem with a UV spectrophotometer on a liquid chromatography system, it is possible to quickly identify the peaks on a UV chromatogram by correlating molecular weight information with each peak - a benefit not available with conventional systems using a UV detector alone. With the unit's PC interface (included in the price of the instrument), researchers can print out the data provided by the detector or download it to a personal computer for data manipulation and printing. Addition of the molecular size detector to a liquid chromatography system provides researchers with a powerful analytical tool that can be used to determine the molecular size, weight, concentration, and purity of proteins as they elute from a column. This eliminates the need for further analysis of column fractions by gel electrophoresis or other methods, resulting in a considerable savings in the time and effort required for most protein purifications. Holly Ahern teaches immunology and cell and molecular biology at the State University of New York, Albany.

Molecular Size Detector Increases Protein Purification

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