This product includes 6 ml of Reagent PMX1 and 6 ml of Reagent PMX2. It is for 200 assays using 96-well plates. Cuvettes may also be used for the assay.
Polymyxins are polypeptide antibiotics that bind lipopolysaccharide (LPS) and disrupt bacterial membranes. They are used for the treatment of Gram-negative bacterial infections. Since polymyxins increase permeability of the bacterial membrane system, they are also used for enhanced release of secreted toxins from bacteria. The MicroGram Polymyxin Assay Kit is designed for measurement of micrograms / ml concentrations of polymyxins. The assay is based on increase of fluorescence at 535 nm of the assay reagent in the presence of polymyxins. The assay kit can be used for measurement of polymyxin concentrations in drug discovery, drug development and pharmaceutical samples.
The MicroGram Polymyxin Assay Kit (Catalog number MPX200) includes 6 ml of Reagent MPX1 and 6 ml of Reagent MPX2. It is for 200 assays using 96-well plates. Cuvettes may also be used for the assay.
ASSAY PROTOCOL
STANDARD CURVE
1. Sample preparation: Prepare 150 µl of polymyxin solutions in the wells of a black 96-well plate with a two-fold serial dilution from 0.2 mg/ml to zero in 90 % methanol.
2. Detection: Mix 30 µl of Reagent MPX1 with 150 µl of the polymyxin solutions for 3 min then add 30 µl of Reagent MPX2 and mix the solution for 15 min. Read the fluorescence at 535 nm (excitation at 485 nm).
3. Data Analysis: Plot the fluorescence intensity Fc and the polymyxin concentration [Polymyxin] to generate the linear standard curve.
Fc = a [Polymyxin] + b
Where the Fc values are from experimental data, the a and b values are from the linear fitting between the Fc values and the polymyxin concentrations.
UNKNOWN SAMPLES
Follow the same procedure to measure the fluorescence intensity Fc values from the unknown samples. Calculate the polymyxin concentrations in the unknown samples using the Fc values from the unknown samples and the a and b values from the standard curve.