Beadlite™ Rapid Colorimetric Maleimide Quantitation Kit for Nanoparticles

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. 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.

1. 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.

1. 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.

The following is a recommended protocol for quantifying the maleimide concentration on the agarose-maleimide sample using a Nanodrop or another UV-vis spectrometer.

1. 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.

2. Set up 3 particle samples: 

   1. Add 100 µL of the maleimide conjugated particles (estimated sample maleimide amount < 20 nmoles).
   2. Centrifuge to remove the supernatant buffer.
   3. Add 400 µL of MES Buffer to resuspend the particles.
   4. Add 100 µL of MEA working solution to particle suspension.
   5. Incubate at room temperature for 20 minutes.

3. Measure absorbance of Total-SH tubes:

   1. Blank the Nanodrop instrument (or other UV-vis spectrometer) with assay buffer (Component C).
   2. Add 10 µL of DTNB Stock Solution to the Total-SH tubes.
   3. 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.
   4. Record the readings, and then calculate their average to obtain the "OD_TSH" value.

4. Measure the absorbance of the sample tubes:

   1. Add 10 µL of DTNB Stock Solution to the sample tubes.
   2. Run the reaction for 2 minutes at room temperature.
   3. Centrifuge the sample tube for 5 minutes (3000 rpm or higher until the supernatant is clear).
   4. Transfer the supernatant to a clean centrifuge tube, then centrifuge for 5 minutes at 5000rpm (or higher) until the supernatant is clear.
   5. Measure A_412nm Nanodrop instrument (or other UV-vis spectrometer).
   6. Record the readings, and then calculate their average to obtain the "OD_Sample" value.

ΔA₄₁₂ = OD_TSH-OD_Sample

Where:

• OD_TSH = Average optical density of the Total SH tubes
• OD_Sample = Average optical density of the sample

MEA consumed (µmoles) =(ΔA₄₁₂ ÷ ε_DTNB) x Sample Volume (µL)

Where:

• ΔA₄₁₂ = Change in absorbance at 412 nm
• ε_DTNB = 14,150 M^-1cm^-1

Maleimide (µmoles Maleimide/mL Beads) = MEA consumed ÷ m

Where:

• m = The volume of the maleimide conjugated sample in mL