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).
Alexa fluor 680 goat anti mouse immunoglobulin g igg
Manufactured by Thermo Fisher Scientific
Alexa Fluor® 680 goat anti-mouse immunoglobulin G (IgG) is a fluorescently labeled secondary antibody. It is designed to detect and bind to mouse primary antibodies, enabling visualization and quantification of target proteins or other biomolecules in various applications such as Western blotting, immunohistochemistry, and flow cytometry.
Automatically generated - may contain errors
Lab products found in correlation
Odyssey infrared imaging system, by LI COR (2 mentions)
Goat anti rabbit igg irdye 800, by Thermo Fisher Scientific (1 mentions)
Image studio lite software, by LI COR (1 mentions)
Odyssey blocking buffer, by LI COR (1 mentions)
Irdye 800cw goat anti mouse igg, by LI COR (1 mentions)
Anti flag m2 monoclonal antibody, by Merck Group (1 mentions)
2 protocols using alexa fluor 680 goat anti mouse immunoglobulin g igg
1
Western Blot Protein Detection Protocol
2
Tricine-SDS-PAGE and Western Blot Analysis
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LPS samples were separated on 14% (w/v) Tricine-SDS-PAGE and the gels stained with silver nitrate (Marolda et al., 2006a) . For Western blot to detect b-O-linked N-acetylglucosamine, samples in the polyacrylamide gel were transferred to nitrocellulose membranes and reacted with O-GlcNAc Monoclonal Antibody (IgM) (Covance) at a 1:1000 dilution. For O-antigen detection anti-O5 antigen monoclonal antibody MF15-4 (Antibodies-online) was used at a 1:10,000 dilution. Alexa Fluor® 680 Goat Anti-Mouse IgM (Invitrogen) or IRDye® 800CW Goat anti-Mouse IgG (LI-COR) were used as secondary antibodies. For protein analysis, polyacrylamide gels were transferred onto nitrocellulose membranes, which were blocked with 10% Western Blocking Solution (Roche Diagnostics). Membranes were incubated overnight at 4 o C with anti-FLAG M2 monoclonal Antibody (Sigma) at a 1:5000 dilution or anti-His (Sigma) at a 1:10,000 dilution. IRDye® 800CW Goat anti-Mouse IgG (LI-COR) or Alexa Fluor® 680 Goat anti-mouse immunoglobulin G (IgG) (Invitrogen) were used as secondary antibodies. Reacting bands were detected by fluorescence with an Odyssey infrared imaging system (Li-cor Bioscience, Lincoln, NE).
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