Beadlite™ Rapid Colorimetric Thiol Quantitation Kit for Nanoparticles

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles

1. Add 250 µL of DMSO (Component C) to dissolve Thiolite™ 490 (Component A).

   Note: Store any unused Thiolite™ 490 stock solution at -20°C (preferably at -80°C), protected from light and moisture. Kit components should remain stable for approximately six months when stored properly.

1. Prepare a 1X Assay Buffer by mixing 1 mL of 10X Assay Buffer (Component B) with 9 mL of ddH₂O.

   Note: 10 mL of 1X Assay Buffer is suitable for 10 samples.

1. Prepare a Thiolite™ 490 working solution by mixing 2 µL of the Thiolite™ 490 stock solution with 200 µL of the 1X Assay Buffer.

   Note: 200 µL is suitable for one assay.

1. Obtain a 100 µL sulfhydryl modified nanoparticle (NP-SH) sample.

2. Centrifuge the NP-SH sample to remove the storage buffer, then wash twice with 100 µL of 1X Assay Buffer per wash.

   Note: When working with magnetic beads, employ a magnetic holder for washing the beads. Avoid using a centrifuge for this step. 

3. After washing, reconstitute the NP-SH sample to a final volume of 100 µL using 1X Assay Buffer.

1. Add 100 µL of the Thiolite™ 490 working solution to the NP-SH sample. Mix thoroughly by either pipetting up and down several times or vortexing briefly for a few seconds.

   Note: The remaining 100 µL of Thiolite™ 490 working solution is used as Total SH.

2. Keep the reaction mixture at room temperature and rotate for 30 to 60 minutes.

1. Measure the absorbance of the Thiolite™ 490 working solution using a Nanodrop (A0).

2. Centrifuge the reaction mixture, and transfer the supernatant into a new clean centrifuge tube.

3. Centrifuge the supernatant at 10,000 rpm for 2 minutes.

4. Transfer the supernatant into a new clean centrifuge tube.

5. Measure the absorbance using a Nanodrop (A1).

   Note: If using a UV-VIS spectrometer to measure the absorbance, dilute the Thiolite™ 490 working solution and the supernatant from steps 1 and 4 of the 'Measure Absorbance' section per the recommended guidelines.

ΔA₄₉₀ = [(A0 x 100) - (A1 x 200)]

Where:

• A0 = Absorbance of Thiolite™ 490 working solution at 490 nm
• A1 = Absorbance of supernatant at 490 nm

µMoles of Maleimide/mg Agarose = [ΔA₄₉₀ ÷ ε_{Thiolite™ 490}] / m

Where:

• ε_{Thiolite™ 490} = 80,000 M^-1cm^-1
• m = weight of sample (mg)