Easy to Perform, Difficult to Optimize for Good Data

October 09, 2015

Grambling State University
Biological Sciences
Assistant Professor

Overall

Quality of Results

Ease-of-Optimization

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Company:

Abcam

Product Name:

Proteasome Activity Assay Kit

Catalog Number:

ab107921

As part of our interest in proteostasis, we have used this kit to measure proteasomal activity. Commercially, there seems to be two approaches, with substrate degradation-based assays being much more common than ELISA. This particular kit uses a popular fluorogenic peptide.

Experimental Design and Results Summary

Application

Assay

Starting Material

Rat hepatoma cell line

Protocol Overview

Lysates and fluorogenic substrate are loaded into a 96 well plate with or without a proteasome inhibitor. The proteasome activity is then based on the difference between those two values.

Tips

Initially, perform a range test and measure frequently to determine the kinetics

Results Summary

Our data (and Abcam's data) shows that the proteasome inhibitor MG132 does not completely block degradation of the substrate. The "window" of proteasome activity likely depends on cell type.

Additional Notes

None

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Summary

The Good

Easy to perform

The Bad

Substrate appears highly susceptible to non-proteasomal cleavage

The Bottom Line

This kit is difficult to optimize especially with conflicting reports in the literature. NP40 is suggested for extraction but some report Triton X100 and NP40 significantly inhibit proteasomes. The calculated activity greatly depends on effective proteasome inhibition by the drug; the HeLa cell data posted on abcam.com is not reassuring.

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