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Preparative Nondenaturing Gel Electrophoresis Used in the Purification of an Esterase Involved in Insecticide Resistance, Rev C

on was determined empirically by analytical, nondenaturing PAGE using the Mini-PROTEAN II cell (Figure 1).

Native PAGE separates proteins both by charge and size and we had protein bands above and below our A2 protein band of interest. At each gel concentration used, the distance was measured between the band of interest and the nearest bands above and below it. The gel concentration was 9%T/3%C where the two curves intersect (Figure 1). The electrode running buffer, in both the upper and the lower buffer chambers, and the buffer used to prepare the gel, was 0.1 M Tris-borate (pH 8.0) containing 2.0 mM EDTA. The sample, after elution from hydroxyapatite, was concentrated using a Centriprep 10 concentrator (Amicon) to approximately 0.5 ml. The sample remained in the elution buffer from the hydroxyapatite column, that is, approximately 60 mM phosphate buffer (pH 6.8). The concentrated sample was mixed with a 5x solution of glycerol and marker dye Xylene Cyanole FF to give a final concentration of 10% glycerol and 0.02% dye marker. The sample, approximately 1.6 mg of protein, was loaded on a 9%T/3%C, 6 cm gel in the Model 491 prep cell (28 mm ID). The Model 491 prep cell had previously been equilibrated overnight in a 4C coldroom. Electrophoresis was performed at 15 W constant power (350400 V). Elution (1 ml/min) and fraction collection (5 ml fractions) began when the marker dye eluted from the gel, after about 1.5 hr run time (Figure 2). The enzyme activity peak was completely eluted about 3.75 hr later. The total run time was 5.5 hr. Electrode buffer was also used as elution buffer because the enzyme is stable in this buffer.


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