Beadlite™ Rapid Colorimetric Maleimide 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 200 µL of distilled water into the vial of MEA (Component A).
Note: 10 µL of the 500X MEA stock solution is enough for 50 reactions (0.5 mL/reaction). Any unused 500X MEA stock solution should be divided into single-use aliquots, stored at -20°C, and kept from light.
Add 1 mL of DMSO (Component D) into the vial of DTNB (Component B) and mix well.
Note: 100 µL of the 50X DTNB stock solution is enough for 10 reactions (0.5 mL/reaction). Any unused 50X DTNB stock solution should be divided into single-use aliquots and stored at -20°C.
PREPARATION OF WORKING SOLUTION
Add 10 µL of MEA stock solution (500X) into 5 mL of distilled water and mix well.
Note: The MEA working solution is not stable. It is recommended to prepare a fresh MEA working solution before use.
SAMPLE EXPERIMENTAL PROTOCOL
The following is a recommended protocol for quantifying the maleimide concentration on the agarose-maleimide sample using a Nanodrop or another UV-vis spectrometer.
Set up 3 Total SH tubes by adding 400µL of Assay Buffer (Component C) and 100 µL of MEA working solution into each tube. Incubate the tubes at room temperature for 20 minutes.
Set up 3 particle samples:
- Add 100 µL of the maleimide conjugated particles (estimated sample maleimide amount < 20 nmoles).
- Centrifuge to remove the supernatant buffer.
- Add 400 µL of MES Buffer to resuspend the particles.
- Add 100 µL of MEA working solution to particle suspension.
- Incubate at room temperature for 20 minutes.
Measure absorbance of Total-SH tubes:
- Blank the Nanodrop instrument (or other UV-vis spectrometer) with assay buffer (Component C).
- Add 10 µL of DTNB Stock Solution to the Total-SH tubes.
- Run the reaction for 2 minutes at room temperature and measure the absorbance of the 3 Total SH tubes at 412 nm without washing the cuvette.
- Record the readings, and then calculate their average to obtain the "ODTSH" value.
Measure the absorbance of the sample tubes:
- Add 10 µL of DTNB Stock Solution to the sample tubes.
- Run the reaction for 2 minutes at room temperature.
- Centrifuge the sample tube for 5 minutes (3000 rpm or higher until the supernatant is clear).
- Transfer the supernatant to a clean centrifuge tube, then centrifuge for 5 minutes at 5000rpm (or higher) until the supernatant is clear.
- Measure A412nm Nanodrop instrument (or other UV-vis spectrometer).
- Record the readings, and then calculate their average to obtain the "ODSample" value.
ΔA412 = ODTSH-ODSample
Where:
- ODTSH = Average optical density of the Total SH tubes
- ODSample = Average optical density of the sample
MEA consumed (µmoles) =(ΔA412 ÷ εDTNB) x Sample Volume (µL)
Where:
- ΔA412 = Change in absorbance at 412 nm
- εDTNB = 14,150 M-1cm-1
Maleimide (µmoles Maleimide/mL Beads) = MEA consumed ÷ m
Where:
- m = The volume of the maleimide conjugated sample in mL
References
Authors: Chen, Haiyan and Zhang, Yiming and Ma, Xiaoxiao and Zhou, Bohao and Liu, Zhonghua
Journal: Biomolecules (2023)
Authors: Olaleye, Oladapo and Spanov, Baubek and Bults, Peter and van der Voort, Anna and Govorukhina, Natalia and Sonke, Gabe S and Horvatovich, Peter and van de Merbel, Nico C and Bischoff, Rainer
Journal: Drug metabolism and disposition: the biological fate of chemicals (2023): 249-256
Authors: Gaiani, Greta and Cucchi, Francesca and Toldrà, Anna and Andree, Karl B and Rey, María and Tsumuraya, Takeshi and O'Sullivan, Ciara K and Diogène, Jorge and Campàs, Mònica
Journal: The Science of the total environment (2022): 150915
Authors: Glenthøj, Andreas and Brieghel, Christian and Nardo-Marino, Amina and van Wijk, Richard and Birgens, Henrik and Petersen, Jesper
Journal: EJHaem (2021): 716-728
Authors: Olaleye, Oladapo and Spanov, Baubek and Ford, Robert and Govorukhina, Natalia and van de Merbel, Nico C and Bischoff, Rainer
Journal: Analytical chemistry (2021): 13597-13605