Beadlite™ Rapid Colorimetric Thiol Quantitation Kit for Nanoparticles
Example protocol
PREPARATION OF STOCK SOLUTIONS
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
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.
PREPARATION OF WORKING SOLUTION
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.
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.
SAMPLE EXPERIMENTAL PROTOCOL
Obtain a 100 µL sulfhydryl modified nanoparticle (NP-SH) sample.
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.
After washing, reconstitute the NP-SH sample to a final volume of 100 µL using 1X Assay Buffer.
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.
Keep the reaction mixture at room temperature and rotate for 30 to 60 minutes.
Measure the absorbance of the Thiolite™ 490 working solution using a Nanodrop (A0).
Centrifuge the reaction mixture, and transfer the supernatant into a new clean centrifuge tube.
Centrifuge the supernatant at 10,000 rpm for 2 minutes.
Transfer the supernatant into a new clean centrifuge tube.
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 ÷ εThiolite™ 490] / m
Where:
- εThiolite™ 490 = 80,000 M-1cm-1
- m = weight of sample (mg)
References
Authors: Kalyani Bhardwaj, Boddapati and Suresh, Padmanaban S
Journal: Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy (2024): 123552
Authors: Weller, Sven and Li, Xin and Petersen, Lars R and Kempen, Paul and Clergeaud, Gael and Andresen, Thomas L
Journal: International immunopharmacology (2024): 111643
Authors: Li, Fayang and Chen, Xianhuang and He, Yuanqiao and Peng, Zhiping
Journal: ACS applied bio materials (2024): 1976-1989
Authors: Sülzle, Jenny and Elfeky, Laila and Manley, Suliana
Journal: Journal of microscopy (2024)
Authors: Hagge, Laurel M and Shahrivarkevishahi, Arezoo and Al-Kharji, Noora M and Chen, Zhuo and Brohlin, Olivia R and Trashi, Ikeda and Tumac, Alisia and C Herbert, Fabian and Adlooru, Abhinay Varma and Lee, Hamilton and Firouzi, Hamid Reza and Cornelius, Samuel A and De Nisco, Nicole J and Gassensmith, Jeremiah J
Journal: Journal of materials chemistry. B (2023): 7126-7133