Neuro2A cells and SH-SY5Y cells were solubilized in lysis buffer (PBS, pH 7.4, 1% n-dodecyl-β-D-maltoside [DDM], 1 mM Na3VO4) containing aprotinin (10 μg/ml), leupeptin (10 μg/ml), and phenylmethylsulfonyl fluoride (1 mM) [22 (link)]. After incubating on ice for 15 min, the lysates were clarified by centrifugation at 12,000 g for 15 min. After protein determination by a Bio-Rad protein assay reagent (Bio-Rad Laboratories, Hercules, CA), the supernatants (20 μg) were subjected to SDS-PAGE and the proteins were transferred to PVDF filter membranes (Millipore, Billerica, MA). The membranes were blocked with 5% non-fat dry milk in Tris-buffered saline containing 0.05% Tween 20 and incubated with primary antibodies. Blots were probed with goat anti-mouse IgG antibody or anti-rabbit IgG antibody coupled to HRP (Bio-Rad Laboratories), and the positive signals were visualized by ECL (PerkinElmer, Waltham, MA). Band intensities were quantified using Image J software.
Anti rabbit igg antibody coupled to hrp
Manufactured by Bio-Rad
The Anti-rabbit IgG antibody coupled to HRP is a laboratory reagent used for the detection of rabbit immunoglobulin G (IgG) in various immunoassays. The antibody is conjugated to the enzyme horseradish peroxidase (HRP), which catalyzes a color-producing reaction when exposed to a suitable substrate. This product can be used as a detection tool in techniques such as Western blotting, ELISA, and other immunochemical methods.
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2 protocols using anti rabbit igg antibody coupled to hrp
1
Solubilization and Immunoblotting of Cell Lysates
2
Western Blot Analysis of Neuronal Proteins
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The neuronal cells (11 DIV) or SH-SY5Y cells were solubilized in lysis buffer (PBS, pH 7.4, 1% n-dodecyl-β-D-maltoside [DDM], 1 mM Na3VO4) containing aprotinin (10 µg/ml), leupeptin (10 µg/ml), and phenylmethylsulfonyl fluoride (1 mM). After incubating on ice for 15 min, the lysates were clarified by centrifugation at 12,000 g for 15 min. After protein determination by a Bio-Rad protein assay reagent (Bio-Rad Laboratories, Hercules, CA), the supernatants (20 µg) were subjected to SDS-PAGE and the proteins were transferred to PVDF filter membranes (Millipore, Billerica, MA). The membranes were blocked with 5% non-fat dry milk in Tris-buffered saline containing 0.05% Tween 20 and incubated with primary antibodies. Blots were probed with goat anti-mouse IgG antibody or anti-rabbit IgG antibody coupled to HRP (Bio-Rad Laboratories), and the positive signals were visualized by ECL (PerkinElmer, Waltham, MA). Band intensities were quantified using Image J software (1.47V, US National Institutes of Health).
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