Dinitrophenylation Assay For Polymixin B

Reference:

Bader, J. and M. Teuber. (1973). Binding action of Polymyxin B on bacterial membranes, to the O-antigenic lipopolysaccharide of S. typhimurium. Z. Naturforsch. 28c:422-430.

Method:

1. Prepare a standard curve (in duplicate) from a 1 mg/ml stock solution of polymyxin B using 5, 10, 20, 30, 40 and 50 ml of the stock solution and all the volumes are brought up to a final volume of 50 µl with H₂0.
2. To 50 µl of sample containing 5-50 µg of polymyxin B, add 200 µl of a 1.0% solution of Na₂B₄O₇.10H₂O in distilled H₂O and 25 µl of 100 mM 1-fluoro 2,4 dinitrobenzene in ethanol.
3. Incubate for 1 hour at 37ºC.
4. Add 1 ml of 2N HCl.
5. Add 1 ml of n-Butanol and vortex.
6. Centrifuge in a clinical centrifuge for 3 minutes at 100 rpm.
7. Read the butanol phase at OD₄₂₀.