NativePAGE

NativePAGE refers to polyacrylamide gel electrophoresis performed under non-denaturing, or native, conditions. This preserves the native conformation of proteins and their interactions, allowing separation by size, shape, and charge without denaturation. As a result, nativePAGE can reveal oligomeric states and protein–protein complexes that are not observable with denaturing methods.

In nativePAGE, samples are prepared in non-denaturing buffers and typically without detergents that disrupt structure or with mild detergents only when needed to solubilize complexes. Reducing agents and detergents that disrupt native interactions are avoided. The gels and running buffers are designed to maintain the proteins’ native state. Variants of nativePAGE include Blue Native PAGE (BN-PAGE), which uses Coomassie Brilliant Blue G-250 to confer charge to proteins and complexes, and Clear Native PAGE (CN-PAGE), which uses no dye. BN-PAGE is especially common for separating membrane protein complexes and large assemblies. A two-dimensional approach combines BN-PAGE with SDS-PAGE to identify subunits after separation.

Applications of nativePAGE include analysis of protein complex composition and stoichiometry, assessment of oligomeric states, studying assembly and disassembly of protein complexes, and evaluating interactions in their near-native state. It can be followed by in-gel activity assays, western blotting, or mass spectrometry for identification. Limitations include reliance on native stability, which can affect resolution and interpretability, and a tendency for mobility to depend on both size and shape, making precise molecular weight estimation more challenging than with denaturing methods.