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At least 721 records · Page 40Linked to original sources
PubMed2001

Identification of nucleotides with identical fluorescent labels based on fluorescence polarization in surfactant solutions.

A solution-phase steady-state polarization-based method for discriminating among the four DNA nucleotides labeled identically with tetramethylrhodamine is described and demonstrated. Labeled nucleotides were dissolved in buffered surfactant solutions. In room temperature 4.5 mM Triton X-100 solutions at neutral pH, the measured steady-state polarizations of tetramethylrhodamine-labeled dATP, dCTP, dGTP and dUTP were 0.261 +/- 0.003, 0.112 +/- 0.003, 0.288 +/- 0.003, and 0.147 +/- 0.003, respectively. A blind test of 40 samples showed no errors in classification based on polarization. The reproducibility obtained during this study demonstrates that the four dye-labeled nucleotides can be discriminated with more than 99.8% confidence.

Deoxyadenine Nucleotides↗
PubMed2003

Kinetics of amyloid beta-protein degradation determined by novel fluorescence- and fluorescence polarization-based assays.

Proteases that degrade the amyloid beta-protein (Abeta) are important regulators of brain Abeta levels in health and in Alzheimer's disease, yet few practical methods exist to study their detailed kinetics. Here, we describe robust and quantitative Abeta degradation assays based on the novel substrate, fluorescein-Abeta-(1-40)-Lys-biotin (FAbetaB). Liquid chromatography/mass spectrometric analysis shows that FAbetaB is hydrolyzed at closely similar sites as wild-type Abeta by neprilysin and insulin-degrading enzyme, the two most widely studied Abeta-degrading proteases. The derivatized peptide is an avid substrate and is suitable for use with biological samples and in high throughput compound screening. The assays we have developed are easily implemented and are particularly useful for the generation of quantitative kinetic data, as we demonstrate by determining the kinetic parameters of FAbetaB degradation by several Abeta-degrading proteases, including plasmin, which has not previously been characterized. The use of these assays should yield additional new insights into the biology of Abeta-degrading proteases and facilitate the identification of activators and inhibitors of such enzymes.

Amino Acid Sequence↗