Blocker casein

Manufactured by Jackson ImmunoResearch

Blocker Casein is a protein-based blocking agent used in immunoassays and Western blotting to reduce non-specific background signals. It is made from purified casein and effectively blocks non-specific protein binding sites on membranes or microplates.

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Lab products found in correlation

Blocker casein, by Thermo Fisher Scientific (2 mentions) Alkaline phosphatase conjugated streptavidin, by Jackson ImmunoResearch (2 mentions) Ez link nhs peg solid phase biotinylation kit, by Thermo Fisher Scientific (2 mentions) Goat anti rabbit igg conjugated to horse radish peroxidase, by Jackson ImmunoResearch (1 mentions) Mouse albumin, by Merck Group (1 mentions)

3 protocols using blocker casein

SARS-CoV-2 NTD Binding Inhibition Assay

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S2X28 mAb was biotinylated using EZ-Link NHS-PEG solid phase biotinylation kit (ThermoFisher Scientific) and binding to NTD tested to set optimal concentration to be used in the assay after sample desalting using Zeba Spin Desalting Columns (ThermoFisher Scietific). Half area 96 well-plates were coated over-night at 4°C with SARS-CoV-2 NTD diluted at 2 μg/mL in PBS. After a blocking step with Blocker Casein (ThermoFisher Scientific), serial plasma dilutions in Blocker Casein were incubated 1 h at room temperature. Biotinylated S2X28 was added at a concentration achieving 70% of maximal binding and the mixture was incubated for 45 min at room temperature. Alkaline-phosphatase conjugated streptavidin (Jackson ImmunoResearch) was diluted at 0.5 μg/mL in Blocker Casein and added on plates previously washed 4 times with PBS 0.05%Tween 20. After 30 min incubation, plates were washed and 4-NitroPhenyl phosphate substrate incubated for 45 min at room temperature. Absorbance at 405 nm was measured and percentage of inhibition was calculated as follows: (1-(OD sample-OD neg ctr)/ (OD pos ctr-OD neg ctr)) x100.

McCallum M., De Marco A., Lempp F.A., Tortorici M.A., Pinto D., Walls A.C., Beltramello M., Chen A., Liu Z., Zatta F., Zepeda S., di Iulio J., Bowen J.E., Montiel-Ruiz M., Zhou J., Rosen L.E., Bianchi S., Guarino B., Fregni C.S., Abdelnabi R., Foo S.Y., Rothlauf P.W., Bloyet L.M., Benigni F., Cameroni E., Neyts J., Riva A., Snell G., Telenti A., Whelan S.P., Virgin H.W., Corti D., Pizzuto M.S, & Veesler D. (2021). N-terminal domain antigenic mapping reveals a site of vulnerability for SARS-CoV-2. Cell, 184(9), 2332-2347.e16.

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Mouse Albumin ELISA Protocol

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Standards (mouse albumin, Sigma) and BAL samples were diluted in borate buffer. After overnight incubation at 4 °C, Blocker™ Casein in PBS (Pierce) was used to inhibit non-specific binding. Rabbit polyclonal antibodies against mouse albumin were allowed to incubate for 1 h (6.9 µg/ml in 10 % Blocker™ Casein in PBS) followed by 1 h incubation of goat anti-rabbit IgG conjugated to horse-radish peroxidase (Jackson ImmunoResearch Laboratories; 1:8000). 3, 3′, 5, 5′ tetramethyl benzidine (TMB) was used as the color reagent and 1.5 N sulfuric acid was used to stop the reaction. The absorbance was read at 465 and 590 nm.

Nemzek J.A., Hodges A.P, & He Y. (2015). Bayesian network analysis of multi-compartmentalized immune responses in a murine model of sepsis and direct lung injury. BMC Research Notes, 8, 516.

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SARS-CoV-2 RBD Binding Inhibition Assay

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S2X259 and S2H14 mAbs were biotinylated using EZ-Link™ NHS-PEG solid phase biotinylation kit (ThermoFisher Scientific) and binding to RBD tested to set optimal concentration to be used in the assay after sample desalting using Zeba ™ Spin Desalting Columns (ThermoFisher Scietific). Half area 96 well-plates were coated over-night at 4°C with SARS-CoV-2 RBD-mouse Fc Tag diluted at 1 μg/ml in PBS. After a blocking step with Blocker Casein (ThermoFisher Scientific), serial plasma dilutions in Blocker Casein were incubated 30 min at room temperature. Biotinylated S2X259 or S2H14 were added at a concentration achieving 80% of maximal binding and the mixture were incubated for 30 min at room temperature. Alkaline-phosphatase conjugated streptavidin (Jackson ImmunoResearch) was diluted at 0.5 μg/ml in Blocker Casein and added on plates previously washed 4 times with PBS 0.05% Tween 20. After 45 min incubation, plates were washed and 4-NitroPhenyl phosphate substrate incubated from 1h at room temperature. Absorbance at 405 nm was measured and percentage of inhibition was calculated as follows: (1-(OD sample-OD neg ctr)/ (OD pos ctr-OD neg ctr)) x100.

Tortorici M.A., Czudnochowski N., Starr T.N., Marzi R., Walls A.C., Zatta F., Bowen J.E., Jaconi S., Di Iulio J., Wang Z., De Marco A., Zepeda S.K., Pinto D., Liu Z., Beltramello M., Bartha I., Housley M.P., Lempp F.A., Rosen L.E., Dellota E J.r., Kaiser H., Montiel-Ruiz M., Zhou J., Addetia A., Guarino B., Culap K., Sprugasci N., Saliba C., Vetti E., Giacchetto-Sasselli I., Fregni C.S., Abdelnabi R., Foo S.Y., Havenar-Daughton C., Schmid M.A., Benigni F., Cameroni E., Neyts J., Telenti A., Virgin H.W., Whelan S.P., Snell G., Bloom J.D., Corti D., Veesler D, & Pizzuto M.S. (2021). Broad sarbecovirus neutralization by a human monoclonal antibody. Nature, 597(7874), 103-108.

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