A Sensitive Fluorimetric Assay for Quantifying Maleimide Groups in Biopolymers

Maleimide groups rapidly and selectively react with thiols, forming stable thioether bonds. This characteristic has led to their widespread use in the formation of stable proteins to proteins or proteins to other biomolecules conjugates. Sensitive assays of maleimide and thiol groups are required for the efficient conjugation of proteins that are expensive and available only in small amounts. The existing methods of quantifying maleimide groups are insensitive and tedious, requiring a large amount sample size. We have developed a sensitive and continuous fluorimetric method for quantifying maleimide groups in proteins, DNA and other biopolymers. This fluorimetric quantitation of maleimide groups is based on Maleimide Green dye that has enhanced fluorescence upon reacting with a maleimide. The method can detect as little as 10 picomoles of maleimide in a 0.1 mL assay volume. It can be performed in a convenient 96‐well or 384‐well microtiter‐plate format and easily adapted to automation with no separation steps required. Its signal can be easily read by a fluorescence microplate reader with Ex/Em = 490/520 nm.