Nitrocellulose membranes are porous membranes composed of nitrocellulose (or, in some commercial formulations, a mixed cellulose ester matrix), used as a solid support for immobilizing proteins, glycoproteins, and nucleic acids. First introduced for protein blotting in 1967, nitrocellulose remains one of the most widely used blotting matrices today.
Properties and Binding Mechanism
Protein binding to nitrocellulose occurs primarily through a combination of hydrophobic and electrostatic interactions, and this binding is described as instantaneous and nearly irreversible, with a quantitative capacity generally cited at 80–100 µg/cm². As one commercial example, Merck's Immobilon-NC membrane, built on a mixed cellulose ester matrix, is offered in a hydrophilic 0.45 µm pore-size format with documented adsorption capacities of approximately 117 µg/cm² for insulin, 160 µg/cm² for BSA, and 259 µg/cm² for goat IgG. Nitrocellulose is easily wetted in water or transfer buffer and is compatible with colorimetric, chemiluminescent, and most fluorescent detection methods. A recognized limitation is that unsupported nitrocellulose is inherently fragile; supported nitrocellulose formats, built on an inert support structure, offer increased strength and can withstand reprobing and autoclaving at 121°C. Nitrocellulose is also soluble in organic solvents such as methanol and acetone, so solvent exposure must be limited to preserve membrane integrity.
Applications
Nitrocellulose membranes are used as the solid support in protein and nucleic acid blotting techniques, including western, northern, and Southern blotting, as well as in colony and plaque lift procedures. In western blotting specifically, they are favored for high binding affinity toward proteins of small-to-mid molecular weight, low background noise, and compatibility with multiple detection methods, generally at a relatively low cost. Compared with PVDF membranes, nitrocellulose is often reported to provide better fluorescence signal-to-noise ratios, while PVDF tends to produce stronger chemiluminescence signals and higher sensitivity for low-abundance proteins. Nitrocellulose is generally considered less suited to nucleic acid transfer applications such as RNA blotting, since the high-salt conditions that favor protein binding can interfere with the charge-based interactions needed for nucleic acid retention.
