Half of the quadriceps muscle was solubilized in 1 ml of tris-buffered saline (TBS) containing 1% Triton X-100 and protease inhibitors. The solubilized fraction was incubated with 200 μl of WGA–agarose bead slurry (Vector Laboratories, Burlingame, CA) overnight at 4°C. Pellets formed from the beads and were washed three times in 1 ml of TBS containing 0.1% Triton X-100 (15 (link)). The beads were then either directly mixed with SDS–polyacrylamide gel electrophoresis (PAGE) loading buffer (for Western blotting, ligand overlay) or eluted with 1 ml of TBS containing 0.1% Triton X-100 and 300 mM N-acetylglucosamine (for solid-phase binding assay). Proteins were separated by 3 to 15% SDS-PAGE and transferred to polyvinylidene difluoride–FL (PVDF-FL; Millipore Sigma) membranes. The membranes were incubated with a sheep polyclonal antibody to human DG (1:100 dilution; R&D Systems, AF6868) and a mouse monoclonal antibody to a glycoepitope on the sugar chain of α-DG (IIH6; 1:100 dilution) followed by IRDye 800CW dye-conjugated goat anti-sheep IgG (LI-COR, 926-32214) and goat anti-mouse IgM (LI-COR, 926-32280), respectively.
Irdye 800cw dye conjugated goat anti sheep igg
Manufactured by LI COR
IRDye 800CW dye-conjugated goat anti-sheep IgG is a secondary antibody that binds to sheep IgG. The dye used is IRDye 800CW, which is a near-infrared fluorescent dye. This product is designed for use in western blotting, immunohistochemistry, and other immunoassay applications that require the detection of sheep IgG.
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2 protocols using irdye 800cw dye conjugated goat anti sheep igg
1
Purification and Detection of Dystroglycan
2
Isolation and Detection of Glycosylated Dystroglycan
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Half of the quadricep muscle was solubilized in 1 mL Tris-buffered saline (TBS) containing 1% Triton X-100 and protease inhibitors. The solubilized fraction was incubated with 200 microliters of WGA-agarose bead slurry (Vector Laboratories, Burlingame, CA) overnight at 4°C. Pellets formed from the beads and were washed three times in 1 mL TBS containing 0.1% Triton X-100 (15) . The beads were then either directly mixed with SDS-polyacrylamide gel electrophoresis (PAGE) loading buffer (for western blotting, ligand overlay) or eluted with 1 mL TBS containing 0.1% Triton X-100 and 300 mM N-acetyl-glucosamine (for solid-phase binding assay). Proteins were separated by 3-15% SDS-PAGE and transferred to polyvinylidene fluoride-FL (PVDF-FL, Millipore Sigma) membranes. The membranes were incubated with a sheep polyclonal antibody to human DG (AF6868; R&D Systems, 1:100 dilution) and a mouse monoclonal antibody to a glycoepitope on the sugar chain of α-DG (IIH6; 1:100 dilution) followed by IRDye® 800CW dye-conjugated goat anti-sheep IgG (LI-COR, 926-32214) and goat anti-mouse IgM (LI-COR, 926-32280), respectively.
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