BDNF was measured in STC-1 lysates via a sandwich ELISA using the Promega Emax immune assay (Promega Corporation, Madison, WI, USA) according to the manufacturer’s protocol. ELISA plates were coated with anti-BDNF mouse antibody (mAb; 1:1000) and incubated overnight at 4°C. The next day the plate was washed and blocked with Blocking Buffer (Promega). BDNF standard or sample (100 μl) was added to each well and incubated for 2 h at room temperature. The plate was washed and anti-BDNF mAb (1:500; 100 μl) was added to each well and incubated for 2 h at room temperature. After washing, 100 μl of diluted anti-IgY HRP (horseradish peroxidase conjugate; 1:200) was added to each well and developed with TMB (3,3’,5,5’-tetramethylbenzidine) solution and 1 N HCl. The absorbance at 450 nm was measured using a VICTOR 2 plate reader and the concentration of BDNF in the samples was calculated from the standard curve and expressed as pg/mg protein [35 (link)]. BDNF assay was repeated 3 times. Each time the samples were run in triplicates containing 100 μl cell lysate from 1-2x106 STC-1 cells/well.
Blocking buffer
Manufactured by Promega
Blocking Buffer is a laboratory reagent used to prevent non-specific binding in various immunoassay techniques, such as Western blotting, ELISA, and immunohistochemistry. It is designed to block unoccupied binding sites on a solid support, helping to reduce background signals and improve the specificity of target detection.
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3 protocols using blocking buffer
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BDNF Quantification in STC-1 Cells
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Quantification of BDNF Protein via ELISA
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BDNF Quantification in Cell Lysates and Media
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BDNF was measured in HBO cells and HEK293 cell lysates and culture medium via a sandwich ELISA using the Promega Emax immune assay (Promega Corporation, Madison, WI, USA) according to the manufacturer’s protocol. ELISA plates were coated with anti-BDNF mouse antibody (mAb; 1:1000) and incubated overnight at 4°C. Next day the plates were washed and blocked with Blocking Buffer (Promega). BDNF standard or sample (100 μl) was added to each well and incubated with shaking for 2h at room temperature. The plates were washed and anti-Human BDNF pAb (1:500; 100 μl) was added to each well and incubated with shaking for 2h at room temperature. After washing, 100 μl of diluted anti-IgY HRP (horseradish peroxidase conjugate; 1:200) was added to each well and developed with TMB (3,3’,5,5’-tetramethylbenzidine) solution. The reaction was stopped by adding 100 μl 1N HCl. The absorbance at 450 nm was measured using a VICTOR 2 plate reader and the concentration of BDNF in the samples was calculated from the standard curve and expressed as pg/2*106 cells as described in detail previously [14 (link), 15 (link)].
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