homogenates

Homogenates are suspensions produced by mechanically disrupting cells or tissues to release intracellular components while keeping soluble constituents in a suitable buffer. They are used to study enzyme activities, protein content, and subcellular processes, and serve as a starting material for fractionation and proteomic analyses.

Preparation typically involves mincing tissue in cold buffer that provides osmotic stabilization, often containing protease inhibitors and mild salts. Mechanical disruption is performed with a pestle and mortar, a Dounce or Potter-Elvehjem homogenizer, or other devices. Buffers commonly use isotonic solutions such as sucrose- or mannitol-based formulations, sometimes with Tris or phosphate buffers; EDTA may be included to chelate divalent cations. After homogenization, the sample is kept on ice to limit proteolysis, and debris are removed by low-speed centrifugation if needed.

Centrifugation is used to separate components by size and density. A typical workflow yields a crude lysate, followed by successive spins to pellet nuclei, then enrich organelles such as mitochondria, lysosomes, and microsomes. Differential centrifugation or density gradient methods may be employed to obtain more refined fractions. The resulting homogenate or fractions are used for enzyme assays, protein quantification, western blotting, subcellular localization studies, and various omics analyses.

Two broad categories exist: tissue homogenates, derived from solid tissues, and cell homogenates, derived from cultured or isolated cells. The term can also refer to aliquots of sample prepared from tissues or cells for experimental use.

Limitations include disruption of organellar integrity, potential proteolysis if not kept cold, and assay interference from debris or detergents. Variability among samples and preparation methods can affect reproducibility.