Several analyses are frequently required to monitor and document the stages of a biomolecule purification process. Electrophoresis is typically used to attest the identity, charge, heterogenicity, consistency and/or purity of products. This powerful analytical technique can also give relevant information about the initial and intermediary materials. The initial or starting material and the component of interest should be fully characterized. Electrophoretic pH-gradient curves, polyacrylamide gel electrophoresis (PAGE), sodium dodecyl sulfate PAGE (SDS/PAGE) and isoelectric focusing are amongst the most useful electrophoresis techniques. Electrophoretic pH-gradient curves of the sample components describe their change in net charge over a given pH interval. This information can guide the choice of proper pH conditions for purification methods which exploit charge differences, and in the choice of gel matrix for ion exchange chromatography. Conventional PAGE resolves proteins according to their size and charge, which is useful for studying the composition and structure of the components. This information can also guide the choice of a gel filtration matrix. SDS/PAGE separates according to subunit molecular weight and is frequently used to ascertain purity, identity, and structure. Isoelectric focusing, which provides high resolution, separates on the basis of isoelectric point (pI) and is capable of separating components differing only slightly in amino acid composition. The choice of technique for detecting the sample components in the gel is important. An automated system allows these previously time-consuming and laborious analyses to be done effectively.
PhastSystemTM is a multitasking workstation for electrophoretic analysis of proteins, peptides, and nucleic acids. It is a semi-automated system that runs up to two gels, under the same conditions within a separation and control unit, while staining up to two gels in a development unit.
Reproducible separations: A wide range of precast PhastGel™ separation media ensures optimal results for isoelectric focusing (IEF), native or SDSPAGE, 2-D electrophoresis and DNA separations. Ready to use PhastGel Buffer Strips for protein or DNA separations, eliminate the need for buffer preparation.
Rapid detection of proteins, peptides, and DNA: PhastSystem offers complete flexibility in the choice of detection method. Ready-to-use reagents and optimized development protocols save time and improve reproducibility. To ensure reliable results, programming instructions are supplied for automated Coomassie™ staining with PhastGel BlueR and silver staining with PhastGel Protein or PhastGel DNA Silver Staining Kits. Other detection methods such as PAS glycoprotein staining, autoradiography or enzyme activity assays can also be used. If required, proteins and peptides can be transferred to immobilization membranes by semi-dry electrophoretic transfer using PhastTransfer Kit, prior to detection.
Ready in minutes for publication: After development, gels are left to air dry or dried quickly (5–10 min) using a hairdryer or microwave oven. PhastGel media do not crack or distort so results are easily stored, ready for publication.
Separations by native or SDS-PAGE: Proteins, peptides or DNA are separated according to size on homogenous or gradient gels. The appropriate gel is selected according to the molecular size range in the sample and the degree of resolution required between the components. Gradient gels are best suited for running unknown samples, giving tight bands, but a compressed pattern. Broad continuous gradients (8–25%) are suitable for separating a wide molecular size range whereas 4–15% is designed for separation of high molecular weight proteins, and 10–15% is designed for the separation of medium molecular weight proteins. Homogenous gels (7.5%, 12.5% and 20%) provide high resolution for a narrow size range. PhastGel Homogenous High Density has been specifically optimized for the separation of smaller molecules such as peptides and oligonucleotides. The same type of gel can be used for native or SDS-PAGE separations. The nature of the separation is determined by selection of the appropriate buffer strips: PhastGel PAGE Buffer Strips, PhastGel SDS-PAGE Buffer Strips or PhastGel DNA Buffer Strips. These ready-to-use, agarose buffer strips contain the buffer required for each separation.