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

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. 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.

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.

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.

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.

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.

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 ÷ ε_{Maleimide 680™}]/m

Where:

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