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Although specific activity gives an indication of purity, additional procedures are necessary to verify that the target protein has been purified to the desired level. Analysis of any mixture to look for contaminants requires a technique with high resolving power. Two procedures offer the kind of sensitivity and resolving power needed for protein analysis and verification, HPLC (High Performance Liquid Chromatography or High Pressure Liquid Chromatography) and polyacrylamide gel electrophoresis (PAGE). HPLC differs from traditional liquid chromatography in that the column bed is made of particles that are extremely small (8-40 microns) and therefore pack together very tightly. Because of the densely packed matrix, high pressures are required to force the sample through the column. The advantages to HPLC include rapid run times and excellent resolution. The disadvantages of HPLC are the expense of the equipment and the high maintenance required to keep it running optimally. Because of this, HPLC systems are not standard equipment in all labs. More sophisticated methods for analysis and verification, such as mass spectrometry, will not be described in the lab manual, as the use of these methods in teaching laboratories is generally cost prohibitive. Consult the additional resources section in Appendix C. Polyacrylamide gel electrophoresis (PAGE) is one of the most powerful methods for protein analysis and is widely available. There are several types of PAGE that are discussed in the textbook (Rosenberg) and resource materials for the course; but in the laboratory, we will concentrate on SDS-PAGE. Determining the degree of purity of a protein preparation is somewhat ambiguous because it depends on the criteria used to define purity. If purification to homogeneity is necessary, the criteria used to specify homogeneity must be defined. For example, if your protein sample is run on SDS PAGE and only one band is present, you might conclude that you had purified the protein to homogeneity. However, two- dimensional SDS PAGE of the same sample could show multiple bands, indicating the presence of multiple proteins with similar molecular weight but with different isoelectric points. For one protein, the purification goal might be a certain specific activity value together with the presence of one band at a particular molecular weight on an SDS PAGE gel. For another protein, the purification goal might be the presence of one band at a certain MW and pI on a 2-D gel. Further confirmation by other methods (such as immunoblotting or HPLC) might also be necessary to ensure that the desired level of purity had been reached. In analyzing our purification of ß-galactosidase, we will compare the number of protein bands present on an SDS PAGE gel, stained with Coomassie Blue, for each of the different stages in the purification procedure and examine how this relates to the specific activity at each point in the purification procedure. Then, in procedure 5.4, immunoblotting will be used to determine which of the protein bands on the gel are actually ß-galactosidase. For each of the steps listed below there is a detailed procedure in this section of your manual:
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