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Maleimide Assays

Sensitive assays of maleimide and thiol groups are required for monitoring the efficient conjugation of proteins that are expensive and available only in small amounts. A variety of crosslinking reagents with a maleimide group are widely used for crosslinking proteins to proteins or proteins to other biomolecules. There are few reagents or assay kits available for quantifying the number of maleimide groups introduced into the first protein.

The overview of Amplite® Rapid Colorimetric Maleimide Quantitation Kit (Cat# 5526).
Maleimides can be directly assayed spectrophotometrically at 302 nm. However, the small extinction coefficient of 620 M-1cm-1 renders the assay insensitive, and the assay is further complicated by the protein absorbance at the same wavelength. Although the enzyme-based maleimide quantification is more sensitive, the method is expensive and extremely time-consuming.

N-ethylmaleimide dose response was measured in a 96-well solid black plate with Amplite® Fluorimetric Maleimide Quantitation Assay Kit using a NOVOstar microplate reader (BMG Labtech).
AAT Bioquest offers the most comprehensive solutions for detecting maleimide group. Table 1. summarizes the features and applications of our maleimide assay kits. These kits have been used for quantifying maleimide groups of different bioconjugates and other materials. Amplite® Rapid Colorimetric Maleimide Quantitation Kit has been specifically optimized for bioconjugates. We strongly recommend you use Amplite® Rapid Colorimetric Maleimide Quantitation Kit for the rapid and accurate quantification of maleimide-containing proteins, oligos and nucleic acids.

Amplite® Rapid Colorimetric Maleimide Quantitation Kit uses our proprietary maleimide sensor Maleimide Blue™ with the maximum absorbance at ~780 nm. The principle of this assay is that Maleimide Blue™ reacts with a maleimide-containing sample, and the resulted product is run through a single spin column to remove the excess sensor. The absorption spectrum of the purified product is measured, and the maleimide to protein ratio can be calculated by the absorbance ratio of 780 nm/280 nm (for proteins) or 780 nm/260 nm (for oligos and nucleic acids). Amplite® Rapid Colorimetric Maleimide Quantitation Kit can be performed using a traditional cuvette spectrophotometer, NanoDrop™ spectrophotometer, or a convenient 96-well absorbance plate reader with a UV-transparent plate.

Amplite® Colorimetric Maleimide Quantitation Assay Kit quantifies a maleimide group by first reacting a sample with a known amount of thiol present in excess and then assaying the remaining unreacted thiol using 4,4'-DTDP with a molar extinction coefficient of 19,800 M-1cm-1. The amount of maleimide is calculated as the difference between the initial amount of thiol and the amount of unreacted thiol after the complete reaction of all maleimide groups. This spectrophotometric assay for the determination of maleimide groups is a reverse GSH assay. It takes advantage of the high reactivity of GSH thiol with the maleimide moiety. Maleimide in the sample is allowed to form a stable thiosuccinimidyl linkage with GSH. After the reaction is complete, the excess GSH, i.e., the remaining thiols of GSH in the reaction mixture, is estimated by using 4,4'-DTDP. The amount of GSH reacted with the sample is titrated to determine the extent of maleimide.

Amplite® Fluorimetric Maleimide Quantitation Kit uses a proprietary dye that has enhanced fluorescence upon reacting with a maleimide. The kit provides a sensitive, one-step fluorimetric method to detect as little as 10 picomoles of maleimide in a 100 µL assay volume (100 nM). The assay can be performed in a convenient 96-well or 384-well microtiter-plate format. Its signal can be easily read using a fluorescence microplate reader at Ex/Em = 490/520 nm. Compared to Amplite® Colorimetric Maleimide Quantitation Assay Kit, this fluorimetric assay is more sensitive, and has less interference from biological samples.

Table 1. Maleimide Quantitation Kits Ordering Information

Cat No.
Product Name
Ex (nm)
Em (nm)
Unit Size
5523Amplite® Fluorimetric Maleimide Quantitation Kit *Green Fluorescence*490515200 Tests
5525Amplite® Colorimetric Maleimide Quantitation Kit324 100 Tests
5526Amplite® Rapid Colorimetric Maleimide Quantitation Kit780 2 Tests