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Blocking buffer

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Blocking buffer is a solution used to prevent non-specific binding in immunoassays, such as ELISA and Western blotting. It contains proteins and detergents that block unoccupied binding sites on the solid support, reducing background signal and improving the specificity of the assay.

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Lab products found in correlation

Facscalibur flow cytometer, by BD (2 mentions) Anti il 4 apc, by Thermo Fisher Scientific (2 mentions) Anti il 17 pe, by Thermo Fisher Scientific (2 mentions) Permeabilization buffer, by Thermo Fisher Scientific (2 mentions) Monensin, by Thermo Fisher Scientific (2 mentions) Fitc anti mouse cd4 rm4 5, by BioLegend (2 mentions) Pacific blue anti mouse cd8 53 6, by Thermo Fisher Scientific (2 mentions) Percp anti ifn γ g1, by Thermo Fisher Scientific (2 mentions) 7 aad viability solution, by BioLegend (2 mentions) Microplate reader, by Thermo Fisher Scientific (1 mentions) 96 well half area elisa plates, by Corning (1 mentions)

Spelling variants of this product

Human Trustain FcX blocking buffer (3 citations) Fc blocking buffer (2 citations)

3 protocols using blocking buffer

1

Flow Cytometric Analysis of Murine Lung Immune Cells

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The left lungs of OVA–induced mice were processed by mincing and then passed through cell strainers. After Percoll density gradient centrifugation, the total number of cells per lung was determined. The single cell suspension was blocked with the blocking buffer (Biolegend, 101320) and stained with various antibodies for flow cytometry analysis (netrophils, CD11b+ Ly-6G+; eosinophils, CD11b+ SiglecF+). For intracelluar cytokine staining, cells were stimulated with phorbol myristate acetate (PMA) (100 ng/mL) and Ionomycin (1 µg/mL) in culture media with monensin (eBioscience, 00-4505-51). After 4 h, cells were washed, blocked with blocking buffer, and stained with the cell surface markers FITC-anti-mouse CD4 (RM4-5, BioLegend, 100510) and Pacific blue-anti-mouse CD8 (53-6.7, eBioscience, 48-0081-82) for 20 min, washed and fixed in 1% paraformaldehyde for 10 min, permeabilized in permeabilization buffer (eBioscience, 00-8333-56) for 5 min, and stained with specific cytokine antibodies APC-anti-IL-4 (11B11, eBioscience, 17-7041-82), PE-anti-IL-17 (ebio17B7, eBioscience, 12-7177-81) and Percp-anti-IFN-γ (G1.2, eBioscience, 45-7311-82) for 20 min. Dead cells were excluded by staining with the 7-AAD viability solution (BioLegend, 420402). Data were acquired on a BD FACS Calibur flow cytometer and analyzed by the FlowJo software.
Qi X., Gurung P., Malireddi R.K., Karmaus P.W., Sharma D., Vogel P., Chi H., Green D.R, & Kanneganti T.D. (2016). Critical role of caspase-8–mediated IL-1 signaling in promoting Th2 responses during asthma pathogenesis. Mucosal immunology, 10(1), 128-138.
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2

Quantification of 146S-Specific Antibody Titers

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The levels of 146S-specific antibody titers, including serum IgG, IgG1, and IgG2a of the immunized mice, were assayed by enzyme-linked immunosorbent assay (ELISA). Briefly, 96-well half-area ELISA plates (Costar, USA) were coated with 5 μg 146S per well and subsequently blocked with blocking buffer (BioLegend, USA). The serum was serially diluted twofold with the blocking buffer. After incubation at 37 °C for 1 hour, HRP-conjugated anti-mouse antibodies (IgM, IgG, IgG1 and IgG2a) were then added to each well at a 1 : 5000 dilution and incubated at room temperature for 1 hour. After washing with PBST, 3,3′,5,5′-tetramethylbenzidine (TMB) solution (Thermo, USA) was added to each plate in the dark for 15 min. Finally, the reactions were stopped by adding 2 M H2SO4. The optical density (OD) was read at 450 nm by using a microplate reader (Thermo, USA). The end-point titers were determined by the maximal serum dilution that exceeded twice the OD values of the control wells.
Wang L., Lin X., Sheng Y., Zhu H., Li Z., Su Z., Yu R, & Zhang S. (2023). Synthesis of a crystalline zeolitic imidazole framework-8 nano-coating on single environment-sensitive viral particles for enhanced immune responses. Nanoscale Advances, 5(5), 1433-1449.
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3

Flow Cytometric Analysis of Murine Lung Immune Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols
The left lungs of OVA–induced mice were processed by mincing and then passed through cell strainers. After Percoll density gradient centrifugation, the total number of cells per lung was determined. The single cell suspension was blocked with the blocking buffer (Biolegend, 101320) and stained with various antibodies for flow cytometry analysis (netrophils, CD11b+ Ly-6G+; eosinophils, CD11b+ SiglecF+). For intracelluar cytokine staining, cells were stimulated with phorbol myristate acetate (PMA) (100 ng/mL) and Ionomycin (1 µg/mL) in culture media with monensin (eBioscience, 00-4505-51). After 4 h, cells were washed, blocked with blocking buffer, and stained with the cell surface markers FITC-anti-mouse CD4 (RM4-5, BioLegend, 100510) and Pacific blue-anti-mouse CD8 (53-6.7, eBioscience, 48-0081-82) for 20 min, washed and fixed in 1% paraformaldehyde for 10 min, permeabilized in permeabilization buffer (eBioscience, 00-8333-56) for 5 min, and stained with specific cytokine antibodies APC-anti-IL-4 (11B11, eBioscience, 17-7041-82), PE-anti-IL-17 (ebio17B7, eBioscience, 12-7177-81) and Percp-anti-IFN-γ (G1.2, eBioscience, 45-7311-82) for 20 min. Dead cells were excluded by staining with the 7-AAD viability solution (BioLegend, 420402). Data were acquired on a BD FACS Calibur flow cytometer and analyzed by the FlowJo software.
Qi X., Gurung P., Malireddi R.K., Karmaus P.W., Sharma D., Vogel P., Chi H., Green D.R, & Kanneganti T.D. (2016). Critical role of caspase-8–mediated IL-1 signaling in promoting Th2 responses during asthma pathogenesis. Mucosal immunology, 10(1), 128-138.
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