DINITROPHENYLATION ASSAY FOR POLYMIXIN B

REFERENCE: Bader, J. and M. Teuber. (1973). Binding action of Polymyxin B on bacterial membranes, I 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 50m l of the stock solution and all the volumes are brought up to a final volume of 50m L with H20.
  2. To 50m l of sample containing 5-50m g of polymyxin B, add 200m l of a 1.0% solution of Na2B4O7.10H2O in distilled H2O and 25m l of 100mM 1-fluoro 2,4 dinitrobenzene in ethanol.
  3. Incubate for 1 hour at 37oC.
  4. Add 1ml of 2N HCl.
  5. Add 1ml of n-Butanol and vortex.
  6. Centrifuge in a clinical centrifuge for 3 minutes at 100rpm.
  7. Read the butanol phase at OD420.