| Description: | The proteasome is a non-lysosome protein degradation system that requires metabolic energy and polymerization of ubiquitin. The 26S proteasome is made up of two subcomplexes (a 20S proteasome and one or two19S regulatory particles). Rapid and gentle isolation of proteasome from cells/tissues is essential for the studies of proteasome structure and function. The proteasomes have been found mainly localized in cytosol but also detected in nucleus. Traditionally, ultracentrifugation and affinity isolation are the most common methods for isolation of proteasomes. These methods, though relatively effective, are usually time consuming with low yield. Many affinity-based methods require harsh elution condition that may affect the activity of isolated proteasomes and limit certain downstream applications. To overcome these shortcomings, we have developed a spin-column based technology using a proprietary precipitation buffer for enrichment of proteasome from cytosol by first removing nuclei and majority of organelles followed by preferential precipitation of cytosolic proteasomes. The nuclear proteasomes are excluded with this kit (Nuclear Proteasome Enrichment Kit is available under Cat # PN-041). The protocol is simple and rapid with high yield. The gentle protocol maintains the association of proteasomes with ubiquitin and other proteins. This kit is useful for the studies of proteasome structure and function. The enriched proteasomes can also be used as a starting material for affinity purification. |
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