Key result
LBTIRFM successfully monitors individual ATP turnovers of single S1dC-GFP fusion proteins in vitro.
Why the study?
A method to assay functions of single protein molecules expressed in vitro is needed, especially for proteins not producible in active form or large quantities by conventional systems.
Population
Single S1dC-GFP fusion proteins expressed in vitro by T7 RNA polymerase and rabbit reticulocyte lysate
Comparison
ATP turnover assay using LBTIRFM vs no assay or conventional methods
Design
Preclinical study using low background total internal reflection fluorescence microscopy
Authors
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May enable single-molecule myosin kinetics studies in cardiomyopathy research; leaves open translation to cellular or clinical cardiac models.
LBTIRFM using GFP as a fluorescent tag allows for the functional assay of single protein molecules expressed in vitro, which is particularly useful for proteins difficult to produce in active form or large quantities.
Iwane et al. (1997) studied this question. Low background total internal reflection fluorescence microscopy (LBTIRFM) was evaluated on Individual ATP turnover. Low background total internal reflection fluorescence microscopy successfully monitored individual ATP turnovers of single S1dC-GFP fusion proteins expressed in vitro.
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