- Make column using Sephadex G-25 in a 50 x 2.5 column. (Bed vol. approx – 100 ml, uses 10-15g dry bead.) Equilibrate column with at least 100 ml of 10mM Na2HPO4 pH7.1/0.145M NaCl buffer.
- Dissolve 40mgs of Polymyxin B sulfate in 1.2 ml of 0.1M NaHCO3.
- Dissolve 10mgs of Dansyl-chloride in 0.8 ml of acetone.
- Add the polymyxin to the Dansyl-chloride and place in the dark for 90 min at room temperature.
- After incubation load the mixture onto a Sephadex G-25 column (50 x 2.5 cm) equilibrated with 10mM Na-phosphate buffer (pH 7.1) containing 0.145M NaCl.
- Collect 5-6ml fractions.
- The Dansyl-Polymyxin comes out in a fairly broad peak ahead of the unreacted Dansyl-chloride peak. Location of the Dansyl-Polymyxin in the collected fractions can be determined by holding a UV lamp over the fractions and looking for fluorescence. The fluorescence of the Dansyl-Polymyxin is yellowish, while the unreacted Dansyl-chloride is more blue-green. The fractions containing the Dansyl-polymyxin are extracted into about 1/2 volume of n-butanol. The butanol is then evaporated to dryness in a glass petri dish place inside a dessicator which is then evacuated and placed at temperature of 37C. This takes about 24 hrs.
- The dried Dansyl-Polymyxin is dissolved in 3 ml of buffer (5mM Hepes, pH 7.0) and stored in aliquots at -20C. The concentration of the Dansyl-polymyxin is determined by dinitrophenylation assay.
NOTE: DPX was commercially available for a few years from Molecular Probes (P13238) in addition to other fluorescently labelled polymxin B compounds, however these products have been discontinued.
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