High-performance liquid chromatography strategy for purifying the transmembrane band 3 protein

Band 3 is the most abundant protein in the red blood cell membrane and has garnered increasing attention across various research fields. However, commercially available recombinant Band 3 is limited to its cytoplasmic domain, typically produced in Escherichia coli, rendering it unsuitable for studies involving the transmembrane region. Furthermore, the purification of Band 3 from red blood cells (RBC) presents challenges due to its strong interactions with membrane and cytoskeletal proteins, often necessitating immunoprecipitation as the primary method to obtain it in a soluble form. This study validates a chromatographic strategy for purifying Band 3 from erythrocyte membranes. Soluble ghosts were processed using reverse-phase chromatography followed by size-exclusion chromatography. Molecular weight validation was performed using polyacrylamide gels, and the presence of soluble Band 3 was confirmed through western blot analysis. This method leverages the physicochemical properties of proteins—specifically their hydrophobicity and size—to facilitate the cost-effective and efficient purification of Band 3.