Taurine Assay Kit

• Reliable Taurine Assay Kit for measurement of free taurine in biological samples.
• Sample taurine concentrations are determined by comparison with a known taurine standard.
• Quick and easy to use.

1. Taurine Standard : One 100 μL tube at 100 mM.
2. 10X Assay Buffer : One 10 mL bottle.
3. 10X Reagent A : One 1 mL tube.
4. 10X Reagent B : One 1 mL amber tube.
5. Cofactor : One amber tube containing 250 mg.
6. Stop Solution : One 5 mL bottle.
7. Quenching Solution : One 5 mL bottle.

Box 2 (shipped on blue ice packs)

• 1X Assay Buffer: Dilute the 10X Assay Buffer to 1X with deionized water. Stir to homogeneity. Store at room temperature.
• 100X Cofactor: Weigh out at least 6 milligrams of solid Cofactor. Resuspend by vortexing thoroughly in 1X Assay Buffer at 6 mg/mL. The 100X Cofactor will change color to bright orange over the first several minutes of resuspension, but is stable for 8 hours at room temperature. Mix 4 100X Cofactor thoroughly just before use in reaction mix below. Discard unused 100X Cofactor after 8 hours at room temperature.
• Reaction Mix: Prepare a Reaction Mix by diluting the 10X Reagent A 1:10, 10X Reagent B 1:10, 100X Cofactor 1:100, and Taurine Dioxygenase 1:10 in 1X Assay Buffer. For example, add 100 μL 10X Reagent A, 100 μL 10X Reagent B, 10 μL 100X Cofactor, and 100 μL of Taurine Dioxygenase to 690 μL of 1X Assay Buffer for a total of 1 mL. This Reaction Mix volume is enough for 20 assays. The Reaction Mix is stable for 1 day at 4 °C. Note: Prepare only enough for immediate use by scaling the above example proportionally.

• Tissue lysates: Sonicate or homogenize tissue sample in cold PBS or 1X Assay Buffer and centrifuge at 10000 x g for 10 minutes at 4 °C. Perform dilutions in 1X Assay Buffer.
• Cell lysates: Resuspend cells at 1-2 x 106 cells/mL in PBS or 1X Assay Buffer. Homogenize or sonicate the cells on ice. Centrifuge to remove debris. Cell lysates may be assayed undiluted or diluted as necessary in 1X Assay Buffer.
• Urine: To remove insoluble particles, centrifuge at 10,000 rpm for 5 min. The supernatant may be assayed undiluted or diluted as necessary in 1X Assay Buffer.
• Serum or Plasma: Deproteinate the sample by running it through a centrifugal filter unit (For example use an Amicon Ultra 0.5 mL 10K Cat. No. UFC501024) and collecting the flow through. To remove insoluble particles, centrifuge at 10,000 rpm for 5 min. The supernatant may be assayed undiluted or diluted as necessary in 1X Assay Buffer. Notes: All samples should be assayed immediately or stored at -80 °C for up to 1-2 months. Run proper controls as necessary. Optimal experimental conditions for samples must be determined by the investigator. Always run a standard curve with samples.

1. Prepare and mix all reagents thoroughly before use. Each sample, including unknowns and standards, should be assayed in duplicate or triplicate. Note: Each sample replicate requires two paired wells, one to be treated with Stop Solution and one with Quenching Solution. While the Stop Solution contains EDTA (which inhibits Taurine Dioxygenase), the Quenching Solution contains both EDTA and a quenching reagent that specifically destroys any sulfite produced by Taurine Dioxygenase, therefore serving as a background control.
2. Add 50 μL of each Taurine Standard or unknown sample into wells of a 96-well microtiter plate.
3. Add 50 μL of Reaction Mix to each well. Mix the well contents thoroughly and incubate for 30 minutes at room temperature.
4. Add 50 μL of Stop Solution to the Taurine Standards and one half of the paired sample wells.
5. Add 50 μL of Quenching Solution to the other half of the paired sample wells.
6. Add 50 μL of Developing Solution to all of the wells and mix thoroughly.
7. Incubate the plate on an orbital shaker for 3 minutes at room temperature.
8. Read the plate at 405-415 nm using a microplate spectrophotometer.

1. Determine the average optical density (OD) values for each sample, control, and standard.
2. Subtract the average zero standard value from itself and all standard values.
3. Graph the standard curve (see Figure 2).
4. Subtract the sample well values containing Quenching Solution from the sample well values containing Stop Solution to obtain the difference. The OD difference is due to the enzyme Taurine Dioxygenase activity (see Figure 3): Net OD = ODStop - ODQuenching
5. Compare the Net OD of each sample to the standard curve to determine and extrapolate the quantity of taurine present in the sample. Only use values within the range of the standard curve.