Journal: Harmful algae

Article Title: Development of a competitive fluorescence-based synaptosome binding assay for brevetoxins

doi: 10.1016/j.hal.2012.06.003

Figure Lengend Snippet: Simple binding of BODIPY®-PbTx-2 (1 nM) was assayed on rat brain synaptosomes. Timing of exposure of rat brain synaptosomes to BODIPY®-PbTx-2 ranged from 5 seconds to 120 minutes to determine binding equilibrium (n = 12). Equilibrium was determined to be achieved in less than 1 hr.

Article Snippet: Curves were analyzed by non-linear regression analysis, and equilibrium inhibition constants (K i ) were determined using the K d value for BODIPY ® -PbTx-2 obtained in saturation experiments, by GraphPad Prism v4.03 using equation 3 : Y = Y min ⁡ + ( Y max ⁡ − Y min ⁡ ) 1 + 10 X − Log EC 50 2.11 Statistical Analysis The specific binding curves for saturation binding were analyzed by non-linear regression analysis by GraphPad Prism v4.03 to compare the fit of the data to one-site and two-site models using the extra sum-of-squares F test.

Techniques: Binding Assay

Journal: Harmful algae

Article Title: Development of a competitive fluorescence-based synaptosome binding assay for brevetoxins

doi: 10.1016/j.hal.2012.06.003

Figure Lengend Snippet: Rat brain synaptosomes were exposed to a range of concentrations of BODIPY®-PbTx-2 (5 nM to less than 0.05 nM). Total binding (without competition) and non-specific binding (with competition from 1 µM PbTx-2) was measured using fluorescence. Specific binding was calculated from the difference between mean total binding and mean non-specific binding at each indicated concentration of BODIPY®-PbTx-2. Representative experiment shown (n = 4 replicates per BODIPY®-PbTx-2 concentration).

Article Snippet: Curves were analyzed by non-linear regression analysis, and equilibrium inhibition constants (K i ) were determined using the K d value for BODIPY ® -PbTx-2 obtained in saturation experiments, by GraphPad Prism v4.03 using equation 3 : Y = Y min ⁡ + ( Y max ⁡ − Y min ⁡ ) 1 + 10 X − Log EC 50 2.11 Statistical Analysis The specific binding curves for saturation binding were analyzed by non-linear regression analysis by GraphPad Prism v4.03 to compare the fit of the data to one-site and two-site models using the extra sum-of-squares F test.

Techniques: Binding Assay, Fluorescence, Concentration Assay

Journal: Harmful algae

Article Title: Development of a competitive fluorescence-based synaptosome binding assay for brevetoxins

doi: 10.1016/j.hal.2012.06.003

Figure Lengend Snippet: Panel A: Competition for binding was assayed using BODIPY®-PbTx-2 (1 nM) against varying concentrations of non-fluorescent PbTx-2 (1 pM to 1 µM) to determine inhibition of BODIPY®-PbTx-2 binding to rat brain synaptosomes. Representative experiment shown (n = 3 replicates per PbTx-2 concentration). Non-specific binding of BODIPY®-PbTx-2 averaged 14.3% +/− 3.2% (n = 5 independent experiments). Panel B: Competitive binding was assayed using BODIPY®-PbTx-2 against varying concentrations of non-fluorescent ligands: PbTx-1, PbTx-2, PbTx-3, and PbTx-9 (1 pM to 10 µM). The thick solid line denotes PbTx-1 binding, the longer hashed line denotes PbTx-2 binding, the shorter hashed line denotes PbTx-3 binding, and the thin solid line denotes PbTx-9 binding (n = 8). Non-specific binding was less than 20% in all cases.

Article Snippet: Curves were analyzed by non-linear regression analysis, and equilibrium inhibition constants (K i ) were determined using the K d value for BODIPY ® -PbTx-2 obtained in saturation experiments, by GraphPad Prism v4.03 using equation 3 : Y = Y min ⁡ + ( Y max ⁡ − Y min ⁡ ) 1 + 10 X − Log EC 50 2.11 Statistical Analysis The specific binding curves for saturation binding were analyzed by non-linear regression analysis by GraphPad Prism v4.03 to compare the fit of the data to one-site and two-site models using the extra sum-of-squares F test.

Techniques: Binding Assay, Inhibition, Concentration Assay