Journal:

Article Title: Resistance of Streptococcus pneumoniae to Deformylase Inhibitors Is Due to Mutations in defB

doi: 10.1128/AAC.45.9.2432-2435.2001

Figure Lengend Snippet: Strains and plasmids used in this work

Article Snippet: pGEX-5X-3 , P tac ; overexpression vector, GST fusion protein , Amersham Pharmacia, Piscataway, N.J..

Techniques: Plasmid Preparation, Expressing, Over Expression, Introduce, Mutagenesis

Journal: Journal of biomolecular screening

Article Title: A Novel High-Throughput Screening Assay for Discovery of Molecules That Increase Cellular Tetrahydrobiopterin

doi: 10.1177/1087057111411088

Figure Lengend Snippet: Design of a time-resolved fluorescence resonance energy transfer (TR-FRET) assay for monitoring the interaction of GTP cyclohydrolase 1 (GTPCH-1) and GTP cyclohydrolase feedback regulatory protein (GFRP). The assay measures the TR-FRET signal upon the binding of a His-tagged GTPCH-1 protein conjugated with a europium–anti-His antibody and glutathione S-transferase (GST)–GFRP conjugated with an allophycocyanin (APC)–anti-GST antibody. Binding of GTPCH-1 and GFRP allows energy transfer from europium to APC and increases the fluorescence emission at 665 nm.

Article Snippet: The human GFRP cDNA was synthesized by GenScript (Piscataway, NJ) and cloned into the pGEX-5X-3 vector (GE Healthcare, Piscataway, NJ) to create the pGEX-5X-3/GFRP construct.

Techniques: Fluorescence, Förster Resonance Energy Transfer, Binding Assay

Journal: Journal of biomolecular screening

Article Title: A Novel High-Throughput Screening Assay for Discovery of Molecules That Increase Cellular Tetrahydrobiopterin

doi: 10.1177/1087057111411088

Figure Lengend Snippet: Optimization of the GTP cyclohydrolase 1 (GTPCH-1)/GTP cyclohydrolase feedback regulatory protein (GFRP) time-resolved fluorescence resonance energy transfer (TR-FRET) binding assay. (A) Effect of titrations of His-GTPCH-1 into solutions of glutathione S-transferase (GST)–GFRP, europium–anti-His, and allophycocyanin (APC)–anti-GST with the necessary cofactors for binding. (B) Effect of titrations of GST-GFRP into solutions of His-GTPCH-1, europium–anti-His, and APC–anti-GST with the necessary cofactors for binding. (C) Z′ factors and signal-to-background (S/B) ratios across the His-GTPCH-1 titration. (D) Z′ factors and S/B ratios across the GST-GFRP titration.

Article Snippet: The human GFRP cDNA was synthesized by GenScript (Piscataway, NJ) and cloned into the pGEX-5X-3 vector (GE Healthcare, Piscataway, NJ) to create the pGEX-5X-3/GFRP construct.

Techniques: Fluorescence, Förster Resonance Energy Transfer, Binding Assay, Titration

Journal: Journal of biomolecular screening

Article Title: A Novel High-Throughput Screening Assay for Discovery of Molecules That Increase Cellular Tetrahydrobiopterin

doi: 10.1177/1087057111411088

Figure Lengend Snippet: Time-resolved fluorescence resonance energy transfer (TR-FRET) assay stability. (A) TR-FRET signals of glutathione S-transferase (GST)–GTP cyclohydrolase feedback regulatory protein (GFRP) titration assay at various time points. (B) Z′ factors across the time course of the assay. (C) Effect of various concentrations of DMSO on the FRET signal using the 384-well and 1536-well plate formats. (D) Z′ factors and signal-to-background (S/B) ratios for assay with increasing DMSO concentrations using the 384-well and 1536-well plate formats.

Article Snippet: The human GFRP cDNA was synthesized by GenScript (Piscataway, NJ) and cloned into the pGEX-5X-3 vector (GE Healthcare, Piscataway, NJ) to create the pGEX-5X-3/GFRP construct.

Techniques: Fluorescence, Förster Resonance Energy Transfer, Titration

Journal: Journal of biomolecular screening

Article Title: A Novel High-Throughput Screening Assay for Discovery of Molecules That Increase Cellular Tetrahydrobiopterin

doi: 10.1177/1087057111411088

Figure Lengend Snippet: Ultra-high-throughput screening (uHTS) format assay validation. The time-resolved fluorescence resonance energy transfer (TR-FRET) assay that monitors GTP cyclohydrolase 1 (GTPCH-1)/GTP cyclohydrolase feedback regulatory protein (GFRP) interaction was validated for uHTS using the Library of Pharmacologically Active Compounds (LOPAC) library in four replicates. Signal-to-background (S/B) ratios (A) and Z′ factors (B) were evaluated for four plates. (C) The percentage of control is calculated as defined in the Materials and Methods section and plotted against compound ID. The potential positives are defined by the compound with % inhibition >30. Representative dose-response curves of the potential positive compounds determined by the TR-FRET assay are shown in panels D, E, and F.

Article Snippet: The human GFRP cDNA was synthesized by GenScript (Piscataway, NJ) and cloned into the pGEX-5X-3 vector (GE Healthcare, Piscataway, NJ) to create the pGEX-5X-3/GFRP construct.

Techniques: High Throughput Screening Assay, Biomarker Discovery, Fluorescence, Förster Resonance Energy Transfer, Control, Inhibition

Journal: Journal of biomolecular screening

Article Title: A Novel High-Throughput Screening Assay for Discovery of Molecules That Increase Cellular Tetrahydrobiopterin

doi: 10.1177/1087057111411088

Figure Lengend Snippet: Validation of positive compounds in the glutathione S-transferase (GST) pull-down assay. GST–GTP cyclohydrolase feedback regulatory protein (GFRP) was bound to glutathione agarose beads, and then His-tagged GTP cyclohydrolase 1 (GTPCH-1) was added. These experiments were performed in the presence of various concentrations of compounds identified by initial high-throughput screening (HTS) of the Library of Pharmacologically Active Compounds (LOPAC) library. The Western blot in the lower panel shows the amount of GFRP used as “bait,” whereas the “trapped” GTPCH-1 is shown in the upper blot. The disappearance of a band in the upper panel indicates disruption of GTPCH/GFRP binding. Panels A, B, and C show the representative GST pull-down assay testing the potential positive compounds. Panels D, E, and F show the representative dose-response curves determined from the densitometry of the GST pull-down assays.

Article Snippet: The human GFRP cDNA was synthesized by GenScript (Piscataway, NJ) and cloned into the pGEX-5X-3 vector (GE Healthcare, Piscataway, NJ) to create the pGEX-5X-3/GFRP construct.

Techniques: Biomarker Discovery, Pull Down Assay, High Throughput Screening Assay, Western Blot, Disruption, Binding Assay