![]() ![]() ![]() Note that the OFFGEL fractionator can be used for conventional in-gel isoelectric focusing using IPG strips. Broad-range (pH 3–10) and narrow-range (pH 4–7) IPG strips are available in both 12- and 24-cm lengths. Current can be controlled for each individual sample. Method programming and execution is performed using a control module consisting of a display screen and keyboard with numerical plus menu and function keys. Two independent power supplies allow simultaneous execution of different methods on the two trays. To accommodate the different frame sizes, the instrument has a fixed connector for the anode electrode and a movable connector for the cathode electrode. For low-resolution separations, 12-well frames are used with 12-cm IPG strips, and for high-resolution separations, 24-well frames are used with 24-cm IPG strips. The trays are positioned on a Peltier-cooled platform. The OFFGEL fractionator can accommodate two trays containing eight samples each for a total of 16 samples. This approach for IEF fractionation reduces the risk of protein precipitation during focusing. At the completion of focusing, proteins diffuse into the well adjacent to the section of the IPG strip within which they have focused. During the separation, sample species migrate through the IPG gel and become focused according to their isoelectric points. Instead of performing focusing in free solution in the presence of carrier ampholytes, proteins and peptides are focused in an immobilized pH gradient (IPG) strip, which is sealed against a multichambered frame containing sample and focusing solutions (Figure 1). The OFFGEL fractionator from Agilent Technologies (Santa Clara, California) uses a novel approach for IEF fractionation. This installment of "Directions in Discovery" will describe four commercial products for isoelectric fractionation of proteins and peptides. Isoelectric fractionation systems are available that perform focusing in solution-phase, off-gel, and in-gel formats. Isoelectric focusing (IEF) has emerged as a useful approach for protein prefractionation because of its high resolution, relatively short separation times, and modest cost (1). Fractionation techniques can include one- or two-dimensional gel electrophoresis, ion-exchange or size-exclusion chromatography, and affinity techniques. IEF Cathode Buffer is used with IEF Anode Buffer in a discontinuous buffer system, which combined with the carrier ampholytes in the gel creates a pH gradient, for focusing proteins within the gel.The enormous complexity of body fluids, cell lysates, or tissue extracts used for proteome analysis necessitates some form of prefractionation before protein identification or quantitative analysis by liquid chromatography (LC)–tandem mass spectrometry (MS) or matrix-assisted laser desorption ionization (MALDI)–MS. Proteins migrate to their isoelectric point (pI), the pH at which the protein has no net charge. IEF separates proteins by their net charge not molecular weight. Use 10x IEF Cathode Buffer for vertical isoelectric focusing (IEF) gels including Ready Gel ® andĬriterion™ IEF Precast Gels. ![]()
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