BDNF Rat ELISA Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
-Allow the microtiter plates to equilibrate to room temperature before opening the foil bags.	-Run a standard curve with each assay. -Drain residual wash liquid from the wells by efficient aspiration or by decantation followed by tapping the plate forcefully on absorbent paper. Never insert absorbent paper directly into the wells. -Do not cover plate with foil.	-Once the desired number of strips are removed, immediately reseal the bag and store the plate at 2 to 8°C to maintain plate integrity. -Store both the concentrate and the Working Wash Buffer in the refrigerator. Use the diluted buffer within 14 days.

Upstream tips
-Allow the microtiter plates to equilibrate to room temperature before opening the foil bags.
Protocol tips
-Run a standard curve with each assay. -Drain residual wash liquid from the wells by efficient aspiration or by decantation followed by tapping the plate forcefully on absorbent paper. Never insert absorbent paper directly into the wells. -Do not cover plate with foil.
Downstream tips
-Once the desired number of strips are removed, immediately reseal the bag and store the plate at 2 to 8°C to maintain plate integrity. -Store both the concentrate and the Working Wash Buffer in the refrigerator. Use the diluted buffer within 14 days.

Publication protocol

Brain-derived neurotrophic factor (BDNF, ERBDNF, Thermo scientific), Vascular endothelial growth factor-A (VEGF-A, RRV00, R&D), Insulin-like growth factor 1 (IGF-1, CSB-E04582r, CUSABIO) and Klotho (CSB-E14958r, CUSABIO) levels were determined by quantitative enzyme-linked immunosorbent assay (ELISA) kits. For this, all reagents and samples were brought to room temperature before use. 50–100 µL of standards and diluted serum samples (Dilution factors: 1x for VEGF, 2x for BDNF, 200x for IGF-1 and 1000x for Klotho; diluted in assay diluent) were added to wells and incubated at room temperature (VEGF and BDNF) or 37 °C (IGF-1 and Klotho) for 2 hours with gentle shaking. Assay diluent served as a zero standard (0 pg/mL) for background subtraction to construct a standard curve. For VEGF and BDNF assays, the solution was then discarded and the wells were washed four times with 400 µL of wash buffer before adding 100 µL of VEGF conjugate solution or BDNF biotinylated antibody and incubating for 1 hour at room temperature. For IGF-1 and Klotho assays, the sample solutions were discarded before directly proceeding (w/o washing) with the addition of a biotin-antibody for 1 hour at room temperature. Wells were washed 4x with buffer. For the VEGF assay, 100 µL of substrate solution was added to each well and incubated for 30 mins at room temperature before adding 50 µL of stop solution. For BDNF, IGF-1 and Klotho assays, an additional step consisted of adding 100 µL HRP-avidin to each well for 60 mins at 37 °C, before washing 5x with buffer. 90–100 µL of TMB substrate was then added in each well for 30 mins before adding the stop solution.

Wells were protected from light at all times after the addition of substrate solution. Optical density of each well was measured using an automated microplate reader (PowerWave 340, Bio-Tek) set to 450 nm (correction wavelength set to 540 nm) within 30 minutes of adding the stop solution. A standard curve was constructed by plotting the mean absorbance for each standard against the concentration to draw a best-fit curve through the points on the graph. If the samples were diluted, the concentration read from the standard curve was then multiplied by the dilution factor.

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Manufacturer protocol

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