Dinitrophenylation Assay For Polymixin B
- 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.
- 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.
- Incubate for 1 hour at 37ºC.
- Add 1 ml of 2N HCl.
- Add 1 ml of n-Butanol and vortex.
- Centrifuge in a clinical centrifuge for 3 minutes at 100 rpm.
- Read the butanol phase at OD420.
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