Proteasome Activity Assay Kit (Fluorometric)

Proteasomes are very large (20S, 26S) protein assemblies found in both the nucleus and cytoplasm of all eucaryotes (and in some procaryotes). They are responsible for the degradation and recycling of proteins which have been previously tagged with ubiquitin. Such tagged proteins are degraded into peptides approximately 7-8 amino acids long which are subsequently further degraded. The 20S assembly is the functional protease structure with chymotrypsin-like, trypsin-like and caspase-like protease activities.

Proteasome Activity Assay Kit provides a simple, reproducible, and sensitive tool of measuring the chymotrypsin-like activity of proteasome in cell lysates samples. In this assay, proteasome cleaves a synthetic specific substrate releasing a fluorophore. The fluorescence signal is measured at Ex/Em = 360/460 nm. The increase in fluorescence intensity is directly proportional to proteasome activity in the sample.

• Microplate reader capable of measuring fluorescence intensity at Ex/Em = 360/460 nm
• Precision pipettes to deliver 2 µL to 1 mL volumes
• Incubator at 37°C
• Tubes to prepare sample dilutions
• 15 mL and 50 mL conical tubes
• MilliQ water or another type of double distilled water (ddH₂O)

1. Add 40 µL of samples, positive control, substrate background to the 96-well microtiter plate.
2. Add 10 µL assay buffer to each well.
3. Add 100 µL of standard to the 96-well microtiter plate.
4. Initiate the reaction by adding 50 µL diluted Proteasome substrate solution to each well. Carefully shake the plate for a few seconds to mix well.
5. Measure fluorescence signal. For kinetic reading: Immediately start measuring fluorescence intensity at Ex/Em=360/460 nm continuously at 37°C and record data every 5 mins. for 30 mins.