Ciprofloxacin is an antibiotic that binds DNA topoisomerases and blocks the DNA replication process in bacteria. The MicroGram Ciprofloxacin Assay Kit is designed for measurement of low micrograms of ciprofloxacin. The assay is based on increase of fluorescence at 470 nm of the assay reagent in the presence of ciprofloxacin. The assay kit can be used for measurement of ciprofloxacin concentrations in drug discovery, drug development and pharmaceutical samples. The assay is not compatible with biological samples containing amino acids or other molecules or buffers with amines.
The MicroGram Ciprofloxacin Assay Kit (Catalog number CIP100) includes 1 ml of 10 x CIP dye, 10 ml of Assay buffer, 10 ml of reagent A. It is for measurement of 100 samples using 96- well plates. Cuvettes may also be used for measurements.
ASSAY PROTOCOL
The following assay protocol is based on assays using a 96-well plate for the measurement. The sample volume is 100 µl and the final assay volume is 200 µl. For assays using cuvette, the sample volume is 500 µl and the final assay volume is 1000 µl.
STANDARD CURVE
1. Sample preparation: Prepare 100 µl of ciprofloxacin in the wells of a black 96-well plate with a two-fold serial dilution from 100 µM to zero in Assay Buffer. For 10 samples, dilute 0.1 ml of the 10 x CIP dye 10-fold with 0.9 ml of Reagent A to make 1 ml of 1 x CIP dye.
2. Detection: Mix 100 µl of 1 x CIP dye with 100 µl of the ciprofloxacin solutions for 5 min and read the fluorescence at 470 nm (excitation at 390 nm).
3. Data Analysis: Plot the fluorescence intensity Fc and the ciprofloxacin concentration [Ciprofloxacin] to generate the linear standard curve.
Fc = a [Ciprofloxacin] + 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 ciprofloxacin concentrations.
UNKNOWN SAMPLES
Follow the same procedure to measure the fluorescence intensity Fc values from the unknown samples. Calculate the ciprofloxacin concentrations in the unknown samples using the Fc values from the unknown samples and the a and b values from the standard curve.