For western blot analysis, proteins from spinal cord tissue (L1–L3 segments) of WT, SMA and SMA+a-C1q mice (n = 2) at P11, were homogenized in lysis buffer (150 mM NaCl, 1% Triton, 2 mM EDTA, 50 mM Tris, pH 7.4) and quantified using Bio-Rad protein assay. Protein extracts (30 μg) were run on 12% SDS–PAGE gels and transferred to PVDF membranes for probing. Blocking was done at room temperature, for 1h, in blocking buffer (5% milk in TBS/0.2% Tween). Membranes were probed with primary antibodies, either mouse monoclonal anti-SMN (1:10000; BD Biosciences, 610646) or mouse monoclonal Anti-α-Tubulin (1:10000; Sigma-Aldrich, T9026) diluted in blocking buffer, and incubated overnight at 4°C. Subsequently, membranes were washed 3 times with TBS/0.2% Tween and incubated for 1 h at room temperature with Peroxidase AffiniPure Goat Anti-Mouse IgG (H+L) (WB, 1:10000; Jackson ImmunoResearch, 115–035-003) secondary antibody diluted in TBS/0.2% Tween. After three sequential 10-min washes, enhanced chemiluminescence (GE Healthcare, Lifesciences) was used for visualization.
Peroxidase affinipure goat anti mouse igg h l wb
Manufactured by Jackson ImmunoResearch
Peroxidase AffiniPure Goat Anti-Mouse IgG (H+L) is a secondary antibody produced in goat and affinity-purified to react with the heavy (H) and light (L) chains of mouse immunoglobulin G (IgG). It is conjugated with horseradish peroxidase (HRP) for use in Western blotting applications.
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2 protocols using peroxidase affinipure goat anti mouse igg h l wb
1
Western Blot Analysis of SMN Protein
2
Western Blot Analysis of SMN Protein
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For western blot analysis, proteins from spinal cord tissue (L1–L3 segments) of WT, SMA and SMA+a-C1q mice (n = 2) at P11, were homogenized in lysis buffer (150 mM NaCl, 1% Triton, 2 mM EDTA, 50 mM Tris, pH 7.4) and quantified using Bio-Rad protein assay. Protein extracts (30 μg) were run on 12% SDS–PAGE gels and transferred to PVDF membranes for probing. Blocking was done at room temperature, for 1h, in blocking buffer (5% milk in TBS/0.2% Tween). Membranes were probed with primary antibodies, either mouse monoclonal anti-SMN (1:10000; BD Biosciences, 610646) or mouse monoclonal Anti-α-Tubulin (1:10000; Sigma-Aldrich, T9026) diluted in blocking buffer, and incubated overnight at 4°C. Subsequently, membranes were washed 3 times with TBS/0.2% Tween and incubated for 1 h at room temperature with Peroxidase AffiniPure Goat Anti-Mouse IgG (H+L) (WB, 1:10000; Jackson ImmunoResearch, 115–035-003) secondary antibody diluted in TBS/0.2% Tween. After three sequential 10-min washes, enhanced chemiluminescence (GE Healthcare, Lifesciences) was used for visualization.
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