Whole cell lysates were resolved by polyacrylamide gel electrophoresis on Nu-Page™ Novex 4-12% bis-Tris gels (for detecting claudin proteins) or Nu-Page™ Novex 3-8% Tris-acetate gels (for ZO-1 proteins) and electrotransferred onto polyvinylidene fluoride membranes (Merck Millipore). Each track was loaded with 15 μg protein, as determined by the Bradford assay (Pierce). Membranes were incubated with pre-titrated primary antibody (Table 1 ) for 16 h at 4°C. Bound antibody was detected with goat anti-mouse IgG IRDye™ 680 or goat anti-rabbit IgG IRDye™ 800 (Invitrogen) and visualised using an Odyssey™ Infra-red Imaging System (Li-Cor Biosciences). Following antibody stripping (Alpha Diagnostic International), blots were re-probed with anti-β-actin antibody for normalisation of protein loading.
Goat anti rabbit igg irdye 800
Manufactured by Thermo Fisher Scientific
Goat anti-rabbit IgG IRDye™ 800 is a secondary antibody used for detecting and quantifying rabbit primary antibodies in various immunoassay applications. It is conjugated with the near-infrared fluorescent dye, IRDye™ 800, which enables sensitive detection and quantification of target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Odyssey infrared imaging system, by LI COR (4 mentions)
Goat anti mouse igg irdye 680, by Thermo Fisher Scientific (2 mentions)
Polyvinylidene fluoride membrane, by Merck Group (2 mentions)
Ecl plus, by GE Healthcare (1 mentions)
Alexa fluor 680 goat anti mouse immunoglobulin g igg, by Thermo Fisher Scientific (1 mentions)
Image studio lite software, by LI COR (1 mentions)
Odyssey blocking buffer, by LI COR (1 mentions)
4 protocols using goat anti rabbit igg irdye 800
1
Western Blot Analysis of Tight Junction Proteins
2
Western Blot Analysis of Tight Junction Proteins
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Whole cell lysates were resolved by polyacrylamide gel electrophoresis on Nu-Page™ Novex 4-12% bis-Tris gels (for detecting claudin proteins) or Nu-Page™ Novex 3-8% Tris-acetate gels (for ZO-1 proteins) and electrotransferred onto polyvinylidene fluoride membranes (Merck Millipore). Each track was loaded with 15 μg protein, as determined by the Bradford assay (Pierce). Membranes were incubated with pre-titrated primary antibody (Table 1 ) for 16 h at 4°C. Bound antibody was detected with goat anti-mouse IgG IRDye™ 680 or goat anti-rabbit IgG IRDye™ 800 (Invitrogen) and visualised using an Odyssey™ Infra-red Imaging System (Li-Cor Biosciences). Following antibody stripping (Alpha Diagnostic International), blots were re-probed with anti-β-actin antibody for normalisation of protein loading.
3
Quantitative Immunoblotting of Mouse and Human Brain Proteins
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Mouse and human brain proteins were electrophoresed on 10% (w/v) SDS-polyacrylamide gels. Insoluble pellets from human brain tissue were resuspended in SDS sample buffer and equivalent amounts were loaded onto gels. Separated proteins were transferred to nitrocellulose membranes, blocked in 5% (w/v) dried skimmed milk in phosphate-buffered saline (PBS) and incubated in primary antibodies (Supplementary Table 2 ), overnight at 4°C. After washing, membranes were incubated with the appropriate fluorophore-conjugated secondary antibody [Alexa Fluor® 680 goat anti-mouse immunoglobulin G (IgG) or IRDye™ 800 goat anti-rabbit IgG, Invitrogen; or horseradish peroxidase-labelled anti-rabbit IgG or anti-mouse IgG, GE Healthcare]. Antigens were visualized using an Odyssey® infrared imaging system (Li-Cor Biosciences) or blots were developed using ECL Plus™ (GE Healthcare) and quantitative analyses were performed using ImageJ software (NIH).
4
Western Blot Protein Detection Protocol
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Proteins in cell lysates and sub-cellular fractions were separated on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Electrophoresed proteins were transferred onto 0.45 μm nitrocellulose membranes. Membranes were blocked in Odyssey blocking buffer (Li-Cor Biosciences), 3% (w/v) dried skimmed milk in Tris-buffered saline/0.2% (v/v) Tween 20 (TBST), or 5% (w/v) bovine serum albumin in TBST for 30 min at ambient temperature, then incubated overnight at 4 °C in primary antibodies (Additional file 1 : Table S1). After washing, membranes were incubated for 60 min at ambient temperature with the appropriate fluorophore-conjugated secondary antibody (Alexa Fluor® 680 goat anti-mouse immunoglobulin G (IgG) or IRDye™ 800 goat anti-rabbit IgG, Invitrogen). Antigens were visualized using an Odyssey® infrared imaging system (Li-Cor Biosciences). Images were analyzed using Li-Cor Image Studio Lite software (Li-Cor Biosciences).
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