Affinity purified Phaseolus vulgaris lectin (PHA-E) is tetrameric glycoprotein and responsible for the erythroagglutinating properties of the PHA fraction. It has a carbohydrate specificity towards oligosaccharides and elutes with bovine thyroglobulin or acetic acid. PHA-E will bind to both human erythrocytes and lymphocytes, with a specificity towards blood group A (-SA). There are five times more PHA-E receptors on normal human lymphocytes than there are on erythrocytes. The crystal structure of a ligand-free PHA-E has a typical legume lectin fold characterized by two anti-parallel β-sheets and two short α-helices, and contains one GlcNAc residue of the N-linked glycan. Asparagine linked erythrocytes glycopeptide is an inhibitor of PHA-E induced agglutination and mitogenicity, and becomes inactive if treated with β-galactosidase. PHA-E binds di-galactosylated and bisected N-glycan. This lectin is widely used as a biochemical tool for detecting bisecting GlcNAc- and Gal-bearing glycoproteins.
Cy5, when bound to Phaseolus vulgaris Lectin (PHA-E), can show the binding pattern of this lectin in cellular imaging and flow cytometry. The excitation wavelength required for Cy5 to fluoresce is high enough to avoid overlap with most other fluorochromes, making it useful for dual-labeling experiments. Because of this high excitation, there is typically less background from autofluorescence of biological specimens.