Synthesis of Dansyl-Polymyxin
Categorized: Outer Membranes
- Dissolve 40mgs of Polymyxin B sulfate in 1.2mls of 0.1M NaHCO3.
- Dissolve 10mgs of Dansyl-chloride in 0.8mls of acetone.
- Add the polymyxin to the Dansyl-chloride and place in the dark for 90 minutes at room temperature.
- After incubation load the mixture onto a Sephadex G-50 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 @ 37oC. This takes about 24hrs.
- The dried Dansyl-Polymyxin is dissolved in 3mls of buffer (5mM Hepes, pH 7.0) and stored in aliquots @ -20oC. The concentration of the Dansyl-polymyxin is determined by dinitrophenylation assay.
- NOTE: To make column buffer, make up 10mM Na2HPO3 and NaH2PO3 plus salt separately and add together to obtain proper pH (about a 1:5 ratio of NaH2PO3 to Na2HPO3).
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