Sulfate plays key roles in metabolism of many important biomolecules including steroids, neurotransmitters, bile acids, glycosaminoglycans and cartilage proteoglycans. Sulfate is also a common ingredient in many pharmaceutical products. The MicroMolar Sulfate Assay Kit (Catalog number MSA200) is designed for measurement of sub-millimolar to millimolar concentrations of sulfate. The assay is based on increase of the light scattering at 600 nm of the sulfate precipitation with Reagent BP. The assay kit can be used for measurements of free sulfate concentrations in biological samples, biochemical reactions, pharmaceutical products and environmental water samples.
The MicroMolar Sulfate Assay Kit (Catalog number MSA200) includes 40 ml of Reagent BP. It is for assays using 96-well plates. Cuvettes may also be used for measurements.
ASSAY PROTOCOL
The following assay protocol is based on using a 96-well plate for the measurement. The sample volume is 100 µl and the final assay volume is 300 µl. For 384-well plate assays, the sample volume is 30 µl and the final assay volume is 90 µl. For assays using cuvette, the sample volume is 333 µl and the final assay volume is 999 µl.
STANDARD CURVE
1. Sample preparation: Prepare 100 µl of sulfate solutions in the wells of a transparent 96-well plate with a two-fold serial dilution from 5 mM to zero in a 10 mM HEPES, pH 7.4 buffer.
2. Detection: Mix 200 µl of Reagent BP with 100 µl of the sulfate solutions for 5 min and read the OD600.
3. Data Analysis: Plot the OD600 and the sulfate concentration [Sulfate] to generate the linear standard curve.
OD600 = a [Sulfate] + b
Where the OD600 values are from experimental data, the a and b values are from the linear fitting between the OD600 values and the sulfate concentrations.
UNKNOWN SAMPLES
Follow the same procedure to measure the OD600 values from the unknown samples. Calculate the sulfate concentrations in the unknown samples using the OD600 values from the unknown samples and the a and b values from the standard curve.