A fluorometric microplate-based assay of submicrogram monomeric actin by inhibition of deoxyribonuclease I.
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 fluorescence 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 dye-DNA complex is sufficient for instrumental detection. With a DNA concentration of 6.0 micrograms/ml and TO dye labeling, as little as 20 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.
Huang
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Molecular Probes, Inc., Eugene, Oregon 97402.
Yue
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You
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Haugland
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RP
D000199:Actins / Q000032:analysis* / Q000494:pharmacology; D000345:Affinity Labels; D000818:Animals; D004247:DNA / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000037:antagonists & inhibitors*; D005456:Fluorescent Dyes; D007700:Kinetics; D008832:Microchemistry / Q000379:methods; D009132:Muscles; D011817:Rabbits; D013050:Spectrometry, Fluorescence / Q000379:methods; D013997:Time Factors
D000199:Actins; D000345:Affinity Labels; D005456:Fluorescent Dyes; D004247:DNA; D003850:Deoxyribonuclease I