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Beadlite™ Rapid Colorimetric Maleimide Quantitation Kit for Nanoparticles *Optimized for 680nm Absorption*

Maleimide-thiol chemistry is integral to the engineering of antibody-nanoparticle (NP) conjugates, with nanoparticle surface activation typically achieved via SMCC-mediated maleimide functionalization. A major challenge in maleimide crosslinking technology is the accurate quantification of maleimide groups covalently attached to solid surfaces. The Beadlite™ Rapid Colorimetric Maleimide Quantitation Kit for Nanoparticles *Optimized for 680nm Absorption* effectively addresses this issue by enabling quick and precise measurements of maleimide functional groups on solid surfaces such as magnetic beads, polystyrene beads, and other nanoparticle types. The kit features Maleimide 680™, which absorbs maximally at 680 nm and is used for the quantitative determination of free sulfhydryl groups. The assay is based on the principle of reacting nanoparticles with an excess of Maleimide 680™, followed by the quantification of unreacted Maleimide 680™ using its known molar extinction coefficient of 250,000 M-1cm-1. The extent of maleimide functionalization on the NP surface is determined by calculating the difference between the initially added and the unreacted Maleimide 680™, allowing for an accurate assessment of maleimide group presence post-reaction. This kit's rapid and straightforward procedure is particularly suitable for applications in bioconjugation research, including the quantification of maleimide functional groups on microspheres and nanoparticles, characterization of maleimide-modified surfaces, and evaluation of maleimide-based crosslinking and conjugation reactions.

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

Maleimide 680™ Stock Solution
  1. 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.

  2. 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

Maleimide 680™ Working Solution
  1. 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

Prepare Sample Solution
  1. Obtain a 100 µL nanoparticle (NP)-maleimide sample.

  2. Centrifuge the NP-maleimide sample to remove the storage buffer and wash with PBS twice (1 mL PBS/wash).

  3. 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.

Run Maleimide Assay
  1. 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.

  2. Keep the reaction mixture at room temperature, gently rotating it for 30 to 60 minutes.
Measure Absorbance
  1. Measure the absorbance of the Maleimide 680™ working solution with 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 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.

DATA ANALYSIS - CALCULATIONS
1. Calculate the change in absorbance

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
2. Calculate the amount of maleimide

µMoles of Maleimide/mg Agarose = [ΔA680 ÷ εMaleimide 680™]/m

Where:

  • ε = 250,000 M-1cm-1
  • m = weight of sample (mg)

References

View all 26 references: Citation Explorer
CRISPR/deadCas9-based high-throughput gene doping analysis (HiGDA): A proof of concept for exogenous human erythropoietin gene doping detection.
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
Antibacterial and hemostatic capacities of cellulose nanocrystalline-reinforced poly(vinyl alcohol) electrospun mats doped with Tiger 17 and pexiganan peptides for prospective wound healing applications.
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
Site-Specific Albumin-Selective Ligation to Human Serum Albumin under Physiological Conditions.
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
Electrochemical biosensor for the dual detection of Gambierdiscus australes and Gambierdiscus excentricus in field samples. First report of G. excentricus in the Balearic Islands.
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
Quantification of Vibrio cholerae cholix exotoxin by sandwich bead-ELISA.
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)
Page updated on June 5, 2025

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Physical properties

Solvent

DMSO

Storage, safety and handling

H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22

Storage

Freeze (< -15 °C); Minimize light exposure
UNSPSC12171501

Components