Amplite® Fluorimetric Maleimide Quantitation Kit *Green Fluorescence*

1. Prepare 20X Maleimide reaction mixture (260 µL)
2. Incubate at room temperature for 30 - 60 minutes
3. Prepare N-ethylmaleimide standards or test samples (50 µL)
4. Add Maleimide working solution (50 µL)
5. Incubate at RT for 5 to 30 minutes
6. Monitor the fluorescence increase at Ex/Em = 490/525 nm (Cutoff = 515 nm)

Thaw all the kit components at room temperature before starting the experiment.

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 20 µL of DMSO (Component E) into the vial of Maleimide Green^TM (Component A) to make 500X Maleimide Green^TM stock solution. Note: 10 µL of 500X Maleimide Green^TM stock solution is enough for one 96-well plate.

Add 10 μL of 500X Maleimide Green^TM stock solution into 250 μL Reaction Buffer (Component B) and mix well to make 20X Maleimide reaction mixture. Incubate 20X Maleimide reaction mixture at room temperature for at least 30 minutes, protected from light. Note: It is very important to incubate the 20X Maleimide reaction mixture for at least 30 mins to maximize the signal to background ratio. Note: You should see the yellow color after adding the 500X Maleimide Green^TM stock solution into Reaction Buffer (Component B).

Add the whole bottle of 20X Maleimide reaction mixture into 5 mL of Assay Buffer (Component C) and mix well to make Maleimide working solution. Note: This Maleimide working solution is not stable. Use within 1 hour.

Table 1. Layout of N-ethylmaleimide standards and test samples in a solid black 96-well microplate. MS = N-ethylmaleimide Standards (MS1 - MS7, 0.14 to 10 µM); BL=Blank Control; TS=Test Samples

Table 2. Reagent composition for each well.

1. Prepare N-ethylmaleimide standards (MS), blank controls (BL), and test samples (TS) according to the layout provided in Tables 1 and 2. For a 384-well plate, use 25 µL of reagent per well instead of 50 µL.
2. Add 50 µL of Maleimide working solution to each well of N-ethylmaleimide standard, blank control and test samples to make the total Maleimide assay volume 100 µL/well. For a 384-well plate, add 25 µL of Maleimide working solution into each well instead, for total volume of 50 µL/well.

3. Incubate the reaction at room temperature for 5 to 30 minutes, protected from light. Note: For best results, the fluorescence intenisty should be read within 30 minutes due to the fact that the fluorescence background increases with time.

4. Monitor the fluorescence increase with a fluorescence microplate reader at Ex/Em = 490/525 nm (Cutoff = 515 nm).