A Sensitive Fluorimetric Assay for Quantifying Maleimide Groups in Biopolymers

Introduction

Maleimide groups rapidly and selectively react with thiols, forming stable thioether bonds. This characteristic has led to its widespread use in the conjugation of proteins to proteins or proteins to other biomolecules. Sensitive assays of maleimide are required to accurately monitor the conjugation of proteins to other biomolecules. In some cases these proteins are expensive and available only in small amounts. The existing methods for quantifying maleimide groups are insensitive and tedious, requiring a large amount sample size.

Fig. 1

Maleimide Green™ Quantifying Assay Principle. The increased in fluorescence signal is proportional to the maleimide concentration in the samples.

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.

Materials and Method

All standard dilutions and HDAC reactions were performed at room temperature with Amplite^® Fluorimetric Maleimide Quantitation Assay Kit (Cat No. 5523).
Standard curve in 50 µL volumes were incubated with equal volumes of maleimide assay mixture in costar 96-solid black well plates for 10 min.
The fluorescence increase was measured as described in Amplite^® Fluorimetric Maleimide Quantitation Assay Kit using a Gemini microplate reader (Molecular Devices) at Ex/Em = 490/525 nm.

Amplite^® Maleimide Assay Workflow

Prepare a 20X maleimide reaction mixture
Incubate at room temperature for 30 to 60 minutes
Prepare maleimide assay mixture (50 µL)
Add maleimide or test samples (50 µL)
Incubate at room temperature for 5 to 30 minutes
Read fluorescence at Ex/Em = 490/525 nm

Maleimide Green™ Spectra

Fig. 2

Excitation and emission spectra of Maleimide Green™ substrate.

Results

Fig. 3

N-ethylmaleimide dose response on 96-well black plate was measured with Amplite^® Fluorimetric Maleimide Quantitation Assay Kit using a Gemini microplate reader at Ex/Em = 490/525 nm.

Fig. 4

The linearity of lower end of N-ethylmaleimide dose response on 96-well black plate was measured with Amplite^® Fluorimetric Maleimide Quantitation Assay Kit using a Gemini microplate reader at Ex/Em = 490/525 nm. As low as 0.1 µM (10 picomol/well) of maleimide can be detected with 10 minutes incubation time (n=3).

Fig. 5

The effect of pH Amplite^® Fluorimetric Maleimide Quantitation Assay Kit. N-ethylmaleimide at 10 mM (A) or 0 mM (Buffer control, B) with differnent pH on 96-well black plate was measured with Amplite^® Fluorimetric Maleimide Quantitation Assay Kit using a Gemini microplate reader at Ex/Em = 490/525 nm.

Summary

Maleimide Green™ substrate is much more sensitive than the traditional methods with the detection limit of 10 picomoles of maleimide in a 0.1 mL assay volume.
The long wavelength emission and higher extinction coefficient of the Maleimide Green™ substrate minimizes less interference from samples.
The Maleimide Green™ Quantitation Assay is a one-step homogeneous assay of the mix and read format.
The assay can be performed in a convenient 96-well or 384-well microtiter-plate format and easily adapted to automation with no separation required.
The Maleimide Green™ Quantitation Assay is a rapid sensitive and robust method for quantifying maleimide groups in proteins and other biopolymers.

Document: 02.0164.200715r1

Last updated Mon Nov 03 2025