A Fluorometric Microplate-Based Assay of Submicrogram Monomeric Actin by Inhibition of Deoxyribonuclease I
✍ Scribed by Z.J. Huang; S. Yue; W.M. You; R.P. Haugland
- Publisher
- Elsevier Science
- Year
- 1993
- Tongue
- English
- Weight
- 496 KB
- Volume
- 214
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
Inhibition of bovine pancreatic deoxyribonuclease I (DNase I) has been a standard method for specific quantitation of monomeric ( (G-) ) actin. The aim of this work is to substantially enhance the sensitivity of this type of G-actin assay by using a fluorescent dye, thiazole orange (TO), which has a high optical absorption, moderate DNA affinity, and large fuorescence enhancement upon binding to DNA. The high fluorescence output and moderate affinity of TO's DNA complex produce a large fluorescence decrease when the complex is disrupted by DNase I, thus permitting a highly sensitive detection of G-actin by its inhibition on the fluorescence decrease. The results show that the dynamic responsiveness of a fluorometric G-actin assay can be improved by lowering the concentration of DNA and affinity of the labeling dye, as long as the fluorescence signal of the dyeDNA complex is sufficient for instrumental detection. With a DNA concentration of (6.0 \mu \mathrm{g} / \mathrm{ml}) and TO dye labeling, as little as (20 \mathrm{ng}) of G-actin can be reliably detected in a fluorescence microplate scanner. This sensitivity also appears to be the utmost detection limit of any G-actin assay that is based on DNase I inhibition. (c) 1993 Academic Press, Inc.