Carfilzomib is an anti-cancer drug. It is a peptide derivative that selectively inhibits the activity of the 20S proteasome. The MicroGram Carfilzomib Assay Kit is designed for measurement of micrograms of carfilzomib. The assay is based on increase of fluorescence at 535 nm of the assay reagent C33 in the presence of carfilzomib. The assay kit can be used for measurement of carfilzomib concentrations in drug discovery and development.
Due to the insolubility of carfilzomib in water, measurement of carfilzomib concentration is performed in DMSO. The assay is not compatible with thiol compounds or metal chelators such EDTA.
The MicroGram Carfilzomib Assay Kit (Catalog number CFZ200) includes 1 ml of 10 x C33 dye for 200 assays using 96-well plates. Cuvettes may also be used for the assay.
ASSAY PROTOCOL
The sample volume is 0.15 ml and the final volume is 0.2 ml in the 96-wellplate assay format.
STANDARD CURVE
1. Dilute 50 µl of 10 x C33 dye 10 fold with ethanol to make 500 µl of 1 x C33 dye.
2. Prepare 150 µl of carfilzomib solutions in DMSO in the wells of a black 96-well plate with a two-fold serial dilution from 0.5 mg/ml to zero in DMSO.
3. Mix 50 µl of 1 x C33 dye with 150 µl of the carfilzomib solutions for 3 min.
4. Read the fluorescence at 535 nm (excitation at 485 nm).
5. Plot the fluorescence intensity Fc and the carfilzomib concentration [Carfilzomib] to generate the linear standard curve.
Fc = a [Carfilzomib] + 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 carfilzomib concentrations.
UNKNOWN SAMPLES
Follow the same procedure to measure the fluorescence intensity Fc values from the unknown samples. Calculate the carfilzomib concentrations in the unknown samples using the Fc values from the unknown samples and the a and b values from the standard curve.