MicroMolar Copper Assay Kit

Mfr.No.	MCA1000
Description	Copper (Cu2+) is an essential metal ion in biological systems. Many enzymes such as amine oxidase and galactose oxidase require copper for their biological functions. Low levels of copper are associated with disorders including mental retardation, depigmentation, anaemia, hypotonia and scorbutic changes in bone. Levels of copper are key diagnostic indicator of diseases such as Wilson's disease, microcytic hypochromic anaemia and bone disease. The MicroMolar Copper Assay Kit is for measurement of micromolar concentrations of free copper ion Cu2+ (0.001 mM – 0.050 mM). The assay is based on the principle that binding the fluorescence dye MCA selectively with copper results in decrease of the fluorescence intensity (emission 535 nm, excitation 485 nm). The assay is compatible with 1 mM Mg2+, Ca2+, Zn2+, Mn2+ and Al3+, 0.1 mM Co2+ and Ni2+. The Cu2+ sample should be diluted in water or 10 mM HEPES buffer, pH 7.4. It is not compatible with Tris buffer. EDTA, thiols, ammonia and amines strongly binds Cu2+ and should be avoided in the assay. The assay kit can be used for high-throughput measurements of copper concentrations in biological samples or environmental water samples. The kit can also be used for biochemical assays of enzyme assays associated with copper metabolism. The kit includes 100 µl of 100 x MCA dye and 30 µl of 1 mM CuCl2. It is for 200 assays using 96- well plates (100 l of sample volume). Cuvettes may also be used for measurements. The kit also provides a 1 mM CuCl2 solution for standard curve.
Concentration measurement	Metal ions

Product Manual	Datasheet
Protocols Description	PROTOCOL The following assay protocol is based on using a 96-well plate the measurement. The sample volume is 100 µl and the final assay volume is 150 µl. For 384-well plate assays, the sample volume is 30 µl and the final assay volume is 45 µl. For assays using cuvette, the sample volume is 660 µl and the final assay volume is 990 µl. STANDARD CURVE 1. Sample preparation: Prepare 100 µl of CuCl2 solutions with a two-fold serial dilution from 0.05 mM to zero in water or a 10 mM HEPES, pH 7.4 buffer. For 10 samples, dilute 7 µl of the 100 x MCA dye 100-fold with water to make 700 µl of 1 x MCA dye. 2. Detection: Mix 100 µl of the CuCl2 sample with 50 µl of the 1x MCA dye solution for 5 min. Read the fluorescence intensity (Fc) at 535 nm (excitation 485 nm). The fluorescence without copper is Fo. 3. Data Analysis: Calculate the Fo / Fc ratio values and plot the correlation between the Fo / Fc ratio values and the copper concentrations to generate the linear standard curve. Fo / Fc = a [Copper] + b Where the Fo / Fc values are from experimental data, the a and b values are from the linear fitting between the Fo / Fc values and the copper concentrations. UNKNOWN SAMPLES Follow the same procedure to get the Fo / Fc ratio values from the unknown samples. Calculate the copper concentrations in the unknown samples using the Fo / Fc ratio values from the unknown samples and the a and b values from the standard curve. [Copper] = (Fo / Fc – b) / a