Beadlite™ Rapid Colorimetric Maleimide Quantitation Kit for Nanoparticles *Optimized for 680nm Absorption*
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
To prepare 1X Assay Buffer, add 1.0 mL of 10X Assay Buffer (Component B) to 9.0 mL of ddH2O.
Note: 10 mL of 1X Assay Buffer is sufficient for 10 tests.
Dissolve Maleimide 680™ (Component A) by adding 40 µL of 1X Assay Buffer.
Note: Store Maleimide 680™ (Component A) in a dark, moisture-free environment at -20 °C, though storing at -80 °C is preferable. When stored under these conditions, the kit components will remain stable for up to six months.
PREPARATION OF WORKING SOLUTION
To prepare the working solution, add 2 µL of Maleimide 680™ stock solution to 200 µL of 1X Assay Buffer, and mix thoroughly.
Note: 200 uL is sufficient for one sample test.
SAMPLE EXPERIMENTAL PROTOCOL
Obtain a 100 µL nanoparticle (NP)-maleimide sample.
Centrifuge the NP-maleimide sample to remove the storage buffer and wash with PBS twice (1 mL PBS/wash).
After washing, reconstitute the NP-maleimide sample to a final volume of 100 μL using 1X Assay Buffer.
Note: When working with magnetic beads, employ a magnetic holder for washing the beads. Avoid using a centrifuge for this step.
Add 100 µL of Maleimide 680™ working solution to the NP-maleimide sample. Mix thoroughly by either pipetting up and down several times or by vortexing briefly for a few seconds.
Measure the absorbance of the Maleimide 680™ working solution with 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 with Nanodrop (A1).
Note: If using a UV-VIS spectrometer to measure the absorbance, dilute the Maleimide 680™ working solution and the supernatant from steps 1 and 4 of the 'Measure Absorbance' section per the recommended guidelines.
ΔA680 = [(A0 x 100) - (A1 x 200)]
Where:
- A0 = Absorbance of Maleimide™ 680 working solution at 680 nm
- A1 = Absorbance of supernatant at 680 nm
µMoles of Maleimide/mg Agarose = [ΔA680 ÷ εMaleimide 680™]/m
Where:
- ε = 250,000 M-1cm-1
- m = weight of sample (mg)
References
Authors: Yi, Joon-Yeop and Kim, Minyoung and Ahn, Jung Ho and Kim, Byung-Gee and Son, Junghyun and Sung, Changmin
Journal: Talanta (2023): 124455
Authors: Teixeira, Marta A and Antunes, Joana C and Seabra, Catarina L and Fertuzinhos, Aureliano and Tohidi, Shafagh D and Reis, Salette and Amorim, M Teresa P and Ferreira, Diana P and Felgueiras, Helena P
Journal: Biomaterials advances (2022): 212830
Authors: Yu, Xingjian and Ruan, Ming and Wang, Yongheng and Nguyen, Audrey and Xiao, Wenwu and Ajena, Yousif and Solano, Lucas N and Liu, Ruiwu and Lam, Kit S
Journal: Bioconjugate chemistry (2022): 2332-2340
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: Awasthi, Sharda Prasad and Chowdhury, Nityananda and Hatanaka, Noritoshi and Hinenoya, Atsushi and Ramamurthy, Thandavarayan and Asakura, Masahiro and Yamasaki, Shinji
Journal: Journal of medical microbiology (2021)