nondenaturing

Nondenaturing refers to experimental conditions that preserve the native structure and interactions of biomolecules, as opposed to denaturing conditions that unfold proteins or nucleic acids. In nondenaturing experiments, common denaturants such as SDS, urea, and extreme pH or heat are avoided, and buffers are chosen to maintain physiological or near-physiological conditions. The goal is to keep quaternary structure, subunit associations, and functional activity intact.

In electrophoresis, nondenaturing (native) methods separate macromolecules without disrupting their folded state. Native PAGE and native agarose gel electrophoresis rely on intact structures and interactions, with migration influenced by size, shape, and charge rather than by complete unfolding. Variants such as blue native PAGE use Coomassie dye to provide charge and enable visualization of protein complexes; clear native PAGE relies on minimal dye effects.

Applications include analysis of oligomeric state, protein–protein and protein–nucleic acid interactions, and assessment of functional activity after separation. In chromatography, non-denaturing (native) conditions are used in techniques such as size-exclusion chromatography and affinity chromatography to preserve complexes during purification.

Considerations include buffer composition, pH, ionic strength, and the presence of mild detergents or nonionic surfactants to solubilize hydrophobic proteins without denaturation. Reducing agents may be avoided if disulfide-linked interactions should be preserved. Limitations include lower resolution for proteins of similar size, potential complex dissociation during handling, and interpretation can be more challenging due to dependence on shape and charge.