ErythroStep™ EMA

ErythoStepTM EMA is based on the selective reactivity of maleimide groups toward free sulfhydryl (—SH) residues present on proteins. Maleimide reacts specifically with accessible thiol groups under near-physiological conditions, forming a stable and irreversible thioether bond. In intact erythrocytes, the number of exposed surface thiols is limited but detectable, primarily originating from transmembrane proteins such as band 3 (anion exchanger 1, AE1), glycophorins, and other membrane-associated proteins whose cysteine accessibility may vary depending on membrane integrity, oxidative status, and storage conditions. Upon incubation with 5-maleimido-eosin, accessible membrane thiols are covalently labeled, anchoring the eosin fluorophore to the erythrocyte membrane.

The eosin moiety is a xanthene-based fluorophore excitable at 488 nm, producing emission in the green-yellow spectrum (~540–570 nm), compatible with standard FITC/PE detection channels depending on optical configuration. Because the labeling is covalent, fluorescence remains stable and does not redistribute between cells. Alterations in erythrocyte membrane protein composition or organization—such as those described in hereditary spherocytosis (HS)—may result in modified EMA fluorescence intensity patterns. For this reason, EMA labeling is widely used in research protocols supporting the evaluation of erythrocyte membrae disorders.