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A new trend to determine biochemical parameters by quantitative FRET assays.

Liao JY, Song Y, Liu Y - Acta Pharmacol. Sin. (2015)

Bottom Line: Historically, FRET assays have been used to quantitatively deduce molecular distances.In this review, we discuss the use of quantitative FRET assays for the determination of biochemical parameters, such as the protein interaction dissociation constant (K(d)), enzymatic velocity (k(cat)) and K(m).We also describe fluorescent microscopy-based quantitative FRET assays for protein interaction affinity determination in cells as well as fluorimeter-based quantitative FRET assays for protein interaction and enzymatic parameter determination in solution.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioengineering, University of California at Riverside, 900 University Avenue, Riverside, CA 92521, USA.

ABSTRACT
Förster resonance energy transfer (FRET) has been widely used in biological and biomedical research because it can determine molecule or particle interactions within a range of 1-10 nm. The sensitivity and efficiency of FRET strongly depend on the distance between the FRET donor and acceptor. Historically, FRET assays have been used to quantitatively deduce molecular distances. However, another major potential application of the FRET assay has not been fully exploited, that is, the use of FRET signals to quantitatively describe molecular interactive events. In this review, we discuss the use of quantitative FRET assays for the determination of biochemical parameters, such as the protein interaction dissociation constant (K(d)), enzymatic velocity (k(cat)) and K(m). We also describe fluorescent microscopy-based quantitative FRET assays for protein interaction affinity determination in cells as well as fluorimeter-based quantitative FRET assays for protein interaction and enzymatic parameter determination in solution.

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Related in: MedlinePlus

Concentrations of digested substrate during pre-SUMO maturation process analyzed by the internal calibration method. Reactions were monitored within the original 5 min.
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fig8: Concentrations of digested substrate during pre-SUMO maturation process analyzed by the internal calibration method. Reactions were monitored within the original 5 min.

Mentions: Pre-SUMO1's maturation by SENP1c can be determined by monitoring the changes in the fluorescent signal at 475 nm and 530 nm in response to excitation at 414 nm during the process. Different amounts of the fluorescent substrates were incubated with SENP1c. The concentration of digested substrate, x, was then calculated according to the quantitative FRET analysis in the internal calibration detection (Figure 8).


A new trend to determine biochemical parameters by quantitative FRET assays.

Liao JY, Song Y, Liu Y - Acta Pharmacol. Sin. (2015)

Concentrations of digested substrate during pre-SUMO maturation process analyzed by the internal calibration method. Reactions were monitored within the original 5 min.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4816234&req=5

fig8: Concentrations of digested substrate during pre-SUMO maturation process analyzed by the internal calibration method. Reactions were monitored within the original 5 min.
Mentions: Pre-SUMO1's maturation by SENP1c can be determined by monitoring the changes in the fluorescent signal at 475 nm and 530 nm in response to excitation at 414 nm during the process. Different amounts of the fluorescent substrates were incubated with SENP1c. The concentration of digested substrate, x, was then calculated according to the quantitative FRET analysis in the internal calibration detection (Figure 8).

Bottom Line: Historically, FRET assays have been used to quantitatively deduce molecular distances.In this review, we discuss the use of quantitative FRET assays for the determination of biochemical parameters, such as the protein interaction dissociation constant (K(d)), enzymatic velocity (k(cat)) and K(m).We also describe fluorescent microscopy-based quantitative FRET assays for protein interaction affinity determination in cells as well as fluorimeter-based quantitative FRET assays for protein interaction and enzymatic parameter determination in solution.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioengineering, University of California at Riverside, 900 University Avenue, Riverside, CA 92521, USA.

ABSTRACT
Förster resonance energy transfer (FRET) has been widely used in biological and biomedical research because it can determine molecule or particle interactions within a range of 1-10 nm. The sensitivity and efficiency of FRET strongly depend on the distance between the FRET donor and acceptor. Historically, FRET assays have been used to quantitatively deduce molecular distances. However, another major potential application of the FRET assay has not been fully exploited, that is, the use of FRET signals to quantitatively describe molecular interactive events. In this review, we discuss the use of quantitative FRET assays for the determination of biochemical parameters, such as the protein interaction dissociation constant (K(d)), enzymatic velocity (k(cat)) and K(m). We also describe fluorescent microscopy-based quantitative FRET assays for protein interaction affinity determination in cells as well as fluorimeter-based quantitative FRET assays for protein interaction and enzymatic parameter determination in solution.

Show MeSH
Related in: MedlinePlus