Separation gels, color detection enzymes

Immediately purify the oxidized enzyme by gel filtration using a column of Sephadex G-25. The chromatography buffer is 0.01 M sodium phosphate, 0.15 M NaCl, pH 7.2. To obtain efficient separation between the oxidized enzyme and excess periodate, the sample size applied to the column should be at a ratio of no more than 5 % sample volume to the total column volume. Collect 0.5-ml fractions and monitor for protein at 280 nm. HRP also may be detected by its absorbance at 403 nm. In oxidizing large quantities of HRP, the fraction collection process may be done visually—just pooling the colored HRP peak as it comes off the column. 

Activity stains are of great importance during the isolation, purification, and characterization of enzymes, since a particular catalytic reaction is involved and the detection of this activity leads to the unequivocal identification of the zone of interest on the electrophoresis gel. Following separation, the gel is removed from the electrophoresis apparatus and is immersed in a minimal volume of a substrate solution. Detection relies on the formation of a colored product by enzyme in the zones containing the enzyme. Examples of activity stains are given in Table 9.2. 

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