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Measurement of proteasome inhibition in live cells in Molecular Devices microplate fluorometers

bition of an uncharacterized protease gsecretase in HEK-293 cells.6


The Z factor is a widely-used parameter to indicate the power of a given assay method to distinguish between negative and positive controls. In general, an assay is considered valid if the is Z factor at the maximal response concentration (EC100) is > 0.5.7 In the present experiment, the Z factor at the EC50 was 0.54-0.66. This Z factor at a concentration lower than the EC100 indicates a powerful assay.


CONCLUSIONS
The proteasome sensor is well-suited for high-throughput screening of candidate modulators of proteasome activity and the assay can easily be run in Molecular Devices microplate fluorometers.


REFERENCES
1. Press release by the Royal Swedish Academy of Sciences, 6 October 2004 (http:// nobelprize.org/chemistry/laureates/2004/ index.html).

2. Glickman, M.H. and Adir, N. (2004) The Proteasome and the delicate balance between destruction and rescue. PLoS Biol 2(1):e13 DOI:10.1371/journal.pbio.0020013.

3. http://www.milennium.com.

4. Clontechniques April 2004 (http://www. bdbiosciences.com/clontech/archive/ APR04UPD/Living_Colors.shtml).

5. Clontechniques April 2003 (http://www. bdbiosciences.com/clontech/archive/ APR03UPD/pdf/ProteasomeSensor.pdf).

6. Zhang, L., Song, L., Parker, E.M. Calpain inhibitor I increases beta-amyloid peptide production by inhibiting the degradation of the substrate of gamma-secretase. Evidence that substrate availability limits beta-amyloid peptide production. J Biol Chem 1999 Mar 26;274(13):8966-72.

7. Zhang, J.H., Chung, T.D., Oldenburg, K.R. A simple statistical parameter for use in evaluation and validation of high throughput sc
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