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Allow the kit reagents to come to room temperature for 30 minutes. Ensure that all samples have reached room temperature and have been diluted as appropriate prior to running them in the kit. Buffer Preparation The Assay Buffer Concentrate should be diluted 1:2 by taking one part of the Concentrate and adding one part of deionized water prior to use. It is stable for up to 3 months when stored at 4 °C. thiostar® thiol Detection reagent Allow the ziploc pouch to warm completely to room temperature prior to opening. Remove the vial of ThioStar Reagent. Add 1.5 mL of the provided DMSO to the vial. Vortex thoroughly. Store any unused reconstituted Detection Reagent at 4 °C in the desiccated pouchand use within 2 months. standard Preparation Label polypropylene test tubes as #1 through #8. Pipet 900 μL of Assay Buffer into tube #1 and 500 μL into tubes #2-#8. Carefully add 100 μL of the standard stock solution to tube #1 and vortex completely. Add 500 μL of tube #1 to tube #2 and vortex completely. Repeat these serial dilutions for tubes #3 through #8. The concentration of N-Acetylcysteine in tubes 1 through 8 will be 10,000, 5,000, 2,500, 1,250, 625, 312.5, 156.25 and 78.125 nM. use all standards within 2 hours of preparation. std 1 std 2 std 3 std 4 std 5 std 6 std 7 std 8 Assay Buffer Volume (μl) 900 500 500 500 500 500 500 500 Addition Stock Std 1 Std 2 Std 3 Std 4 Std 5 Std 6 Std 7 Volume of Addition (μl) 100 500 500 500 500 500 500 500 Final Conc. (nM) 10,000 5,000 2,500 1,250 625 312.5 156.25 78.125 ® 8 EXPECT ASSAY ARTISTRY
We recommend that all standards and samples be run in duplicate to allow the end user to accurately determine thiol concentrations accurately. 1. A plate layout sheet has been included in the insert on the back page of the insert to aid proper sample and standard identification. Set plate parameters for a 96-well Corning Costar 3686 plate. See www.arborassays.com/resources/#general-info for plate dimension data. 2. Pipet 100 μL of samples, Assay Buffer as the blank or standards into wells in the black plate. 3. Add 25 μL of the ThioStar Reagent to each well using a repeater or multichannel pipet. 4. Gently tap the sides of the plate to ensure adequate mixing of the reagents. 5. Cover the plate with the plate sealer and incubate at room temperature for 30 minutes in the dark. 6. Set plate parameters for a 96-well Corning Costar 3686 plate. See www.arborassays.com/ resources/#general-info for plate dimension data. Read the fluorescent signal from each well in a plate reader capable of reading the fluorescent emission at 510 nm with excitation at 370- 410 nm. Please contact your plate reader manufacturer for suitable filter sets. 7. Use the plate reader's built-in 4PLC software capabilities to calculate thiol concentrations for each sample.
Average the duplicate FLU readings for each standard and sample. Create a standard curve by reducing the data using the 4PLC fitting routine on the plate reader, after subtracting the mean FLUs for the zero standard. The sample concentrations obtained should be multiplied by the dilution factor to obtain neat sample values.