DINITROPHENYLATION ASSAY FOR POLYMIXIN B
Bader, J. and M. Teuber. (1973). Binding action of Polymyxin B on bacterial membranes, I to the O-antigenic lipopolysaccharide of
Prepare a standard curve (in duplicate) from a 1 mg/ml stock solution of polymyxin B using 5, 10, 20, 30, 40 and 50
l of the stock solution and all the volumes are brought up to a final volume of 50
L with H
l of sample containing 5-50
g of polymyxin B, add 200
l of a 1.0% solution of Na
O in distilled H
O and 25
l of 100mM 1-fluoro 2,4 dinitrobenzene in ethanol.
Incubate for 1 hour at 37
Add 1ml of 2N HCl.
Add 1ml of n-Butanol and vortex.
Centrifuge in a clinical centrifuge for 3 minutes at 100rpm.
Read the butanol phase at OD