20S Proteasome Activity Kit

Additional Information

Assay Background

Proteasomes are non-lysosomal proteolytic complexes localised primarily in the cytoplasm and in the nucleus of eukaryotic cells. They are responsible for the ubiquitin-mediated degradation of short half-life proteins and peptides that are involved in essential cellular processes including cell-cycle regulation, apoptosis and transcriptional regulation, innate immunity and antigen processing, and in the removal of redundant or damaged proteins. As such protein degradation by the ubiquitin-proteasome pathway has a major regulatory function for proliferation activity and survival of both normal and malignant cells, and its dysfunction has been implicated in a wide range of other disease processes including neurodegenerative, cardiovascular and metabolic disorders. The 26S proteasome structure is composed of a 20S proteasome catalytic core complex and one or two 19S (PA700) regulatory subcomplexes. The 20S core comprises two copies of 14 subunits (7 alpha subunits and 7 beta subunits) arranged in a α7β7β7α7 cylindrical array. Proteolytic activities are determined by the β1 (caspase-like), β2 (trypsin-like) and β5 (chymotrypsin-like) subunits, access to which is guarded by the α-subunits. The 19S regulatory unit consists of six ATPase and at least ten non-ATPase subunits that are required for ubiquitinated protein binding, deubiquitination, substrate unfolding and translocation to the 20S catalytic core. Varying catalytic subunit composition (β1, β1i; β2, β2i; β5, β5i) results in a variety of possible subtypes from full constitutive proteasome (β1, β2, β5) through mixed populations to full inducible / immunoproteasome (β1i, β2i, β5i). Alternative regulatory complexes such as the PA200 and 11S proteasome activators confer different substrate specificities and activity compared to the 19S regulator.

Assay Principle

The 20S Proteasome Activity kit facilitates the rapid, robust measurement of proteasome activity in vitro. The kit utilises high purity, fluorogenic substrate Suc-LLVY-AMC together with suitable calibration standards and controls for the accurate and sensitive assessment of proteasome chymotrypsin-like (β5) activity. Continuous kinetic or end-point assays can be performed in micro-cuvettes or in 96-well plate format for multi-sample analysis. Contains sufficient materials for one full 96-well plate assay or up to 100x 50µL micro-cuvette based assays to be run.

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