Protein Interactions

ABSTRACT

This protocol describes a method for chemical cross-linking of proteins using formaldehyde. With the exception of zero-length cross-linkers, formaldehyde has the shortest cross-linking span (~2-3 Å) of any cross-linking reagent, thus making it an ideal tool for detecting specific protein-protein interactions with great confidence. Despite its simple chemical structure (CH₂O), formaldehyde’s single carbonyl group functions essentially as a homobifunctional reagent and is capable of conjugating targets through two different chemical pathways. In Mannich-type reactions, which typically require elevated temperatures (37 °C or above) for a period of 2-24 hours for acceptable yields, formaldehyde condenses with amines (1°, 2°, or salts of ammonia) and compounds with active hydrogens to form stable cross-links. Formaldehyde also reacts with amines to form highly reactive immonium cations that are reactive toward protein-containing nucleophiles, including sulfhydryls, amines, phenols, and imidazoles. The latter reaction is more rapid than the former and is generally the more predominant in typical protein cross-linking reactions at ambient temperatures.

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