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Horseradish peroxidase hrp conjugated anti goat or rabbit igg

Manufactured by Santa Cruz Biotechnology

Horseradish peroxidase (HRP)-conjugated anti-goat or rabbit IgG is a secondary antibody that recognizes and binds to the constant region of goat or rabbit immunoglobulin G (IgG). The HRP enzyme attached to the antibody can be used to detect and visualize the target antigen in various immunoassays, such as Western blotting, ELISA, and immunohistochemistry.

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6 protocols using horseradish peroxidase hrp conjugated anti goat or rabbit igg

1

Western Blot Analysis of KEAP1 Protein

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Experimental cells were collected, homogenized, and lysed with lysis buffer (Cell Signaling Technology, Boston, MA, USA) containing 0.5 mM of phenylmethanesulfonyl fluoride (PMSF) (Sigma-Aldrich, St. Louis, MO, USA). Protein concentration was determined by BCA protein assay (Thermo Fisher Scientific, Grand Island, NY, USA). Samples of 25 μg protein were fractionated by SDS-PAGE in 4–20% gradient Tris-glycine precast gels (Invitrogen) and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA). The membrane was incubated for 1 hour in blocking solution containing 5% nonfat milk powder and 0.1% Tween-20, pH 7.6. This was followed by overnight incubation at 4°C in blocking solution containing rabbit primary antibodies against KEAP1 (D6B12, Cell Signaling Technology, Boston, MA, USA). Subsequently, the labeled proteins were visualized by incubation with a horseradish peroxidase- (HRP-) conjugated anti-goat or rabbit IgG (1 : 2000; Santa Cruz Biotechnology, Inc.) followed by development with a chemiluminescence substrate for HRP (Thermo Fisher Scientific). The images of western blots were captured by GE ImageQuant.
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2

Retinal Cofilin Signaling Protein Analysis

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Retinas were lysed with lysis buffer (Cell Signaling Technology, Boston, MA, USA) containing 0.5 mM of phenylmethanesulfonyl fluoride (PMSF) (Sigma-Aldrich, St. Louis, MO, USA). Protein concentration was determined by BCA protein assay (Thermo Fisher Scientific). Samples (25 µg) were separated by SDS-PAGE in 4–20% gradient Tris-glycine precast gels (Invitrogen) and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA). The membrane was incubated for 1 hour in blocking solution containing 5% non-fat milk powder and 0.1% Tween-20, pH 7.6. This was followed by overnight incubation at 4°C in the blocking buffer containing rabbit primary antibodies against cofilin, phospho-cofilin, and phospho-LIMK (all from Cell Signing Technology). Subsequently, the labeled proteins were visualized by incubation with a horseradish peroxidase (HRP)-conjugated anti-goat or rabbit IgG (1:2000; Santa Cruz Biotechnology) followed by development with a chemiluminescence substrate for HRP (Thermo Scientific). The images of western blots were captured by GE imageQuant. Relative band intensities were analyzed using Image J software and normalized to non-phospho-blots and to GAPDH.
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3

Protein Assessment via Cell Extraction

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Cell extractions for protein assessment studies were performed as previously described [12 (link)]. Briefly, cells were lysed with Cell Lysis Buffer (#9803, Cell Signaling Technology, Boston, MA, USA) containing 0.5 mM of phenylmethanesulfonyl fluoride (PMSF) (Sigma-Aldrich, St. Louis, MO, USA). Protein concentration was standardized by BCA protein assay (Thermo Fisher Scientific, Grand Island, NY, USA). Samples (25 μg) were separated by SDS-PAGE in 4% – 20% gradient Tris-glycine precast gels (Invitrogen, Carlsbad, CA, USA) and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA). The membrane was incubated for 1 hour in blocking solution containing 5% non-fat milk powder and 0.1% Tween-20, pH 7.6. This was followed by overnight incubation at 4 C in the blocking buffer containing rabbit primary antibodies against CD36 (Abcam, ab133625, 1:500). Subsequently, the labeled proteins were visualized by incubation with a horseradish peroxidase (HRP)-conjugated anti-goat or rabbit IgG (1:2000; Santa Cruz Biotechnology) followed by development with a chemiluminescence substrate for HRP (Thermo Fisher Scientific). The images of western blots were captured by GE imageQuant imager. Relative band intensities were analyzed using Image J software and normalized to GAPDH.
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4

Western Blot Analysis of KLF4 in Mice

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Adult mice of indicated genotypes were killed, and retinas were dissected and lysed with lysis buffer (Cell Signaling Technology) containing 0.5 mm phenylmethylsulfonyl fluoride (Sigma-Aldrich). Protein concentration was determined by BCA protein assay (Thermo Fisher Scientific). Samples (25 μg) were separated by SDS-PAGE in 4-20% gradient Tris-glycine precast gels (Invitrogen) and transferred to a polyvinylidene difluoride membrane (Millipore). The membrane was incubated for 1 h in blocking solution containing 5% nonfat milk powder and 0.1% Tween-20, pH 7.6. This was followed by overnight incubation at 4°C in the blocking buffer containing rabbit primary antibodies against KLF4 (1:50; Ab72543, Abcam). Subsequently, the labeled proteins were visualized by incubation with a horseradish peroxidase (HRP)-conjugated anti-goat or rabbit IgG (1:2000; Santa Cruz Biotechnology) followed by development with a chemiluminescence substrate for HRP (Thermo Fisher Scientific). The images of Western blots were captured by GE ImageQuant. Relative band intensities were analyzed using ImageJ software and normalized to GAPDH.
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5

KEAP1 Expression in Post-cardiac Arrest Myocardium

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Experimental rats were sacrificed 12 hours after ROSC. Then, the rat hearts were harvested and the left ventricular myocardial tissue was collected, homogenized, and lysed with lysis buffer (Cell Signaling Technology, Boston, MA, USA) containing 0.5 mM of phenylmethanesulfonyl fluoride (PMSF) (Sigma-Aldrich, St. Louis, MO, USA). Protein concentration was determined by BCA protein assay (Thermo Fisher Scientific, Grand Island, NY, USA). Samples of 25 μg protein were fractionated by SDS-PAGE in 4–20% gradient Tris-glycine precast gels (Invitrogen) and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA). The membrane was incubated for 1 hour in blocking solution containing 5% nonfat milk powder and 0.1% Tween-20, pH 7.6. This was followed by an overnight incubation at 4°C in blocking solution containing rabbit primary antibodies against KEAP1 (D6B12, Cell Signaling Technology, Boston, MA, USA). Subsequently, the labeled proteins were visualized by incubation with a horseradish peroxidase- (HRP-) conjugated anti-goat or rabbit IgG (1 : 2000; Santa Cruz Biotechnology) followed by development with a chemiluminescence substrate for HRP (Thermo Fisher Scientific). The images of Western blots were captured by GE imageQuant.
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6

Protein Assessment via Cell Extraction

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Cell extractions for protein assessment studies were performed as previously described [12 (link)]. Briefly, cells were lysed with Cell Lysis Buffer (#9803, Cell Signaling Technology, Boston, MA, USA) containing 0.5 mM of phenylmethanesulfonyl fluoride (PMSF) (Sigma-Aldrich, St. Louis, MO, USA). Protein concentration was standardized by BCA protein assay (Thermo Fisher Scientific, Grand Island, NY, USA). Samples (25 μg) were separated by SDS-PAGE in 4% – 20% gradient Tris-glycine precast gels (Invitrogen, Carlsbad, CA, USA) and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA). The membrane was incubated for 1 hour in blocking solution containing 5% non-fat milk powder and 0.1% Tween-20, pH 7.6. This was followed by overnight incubation at 4 C in the blocking buffer containing rabbit primary antibodies against CD36 (Abcam, ab133625, 1:500). Subsequently, the labeled proteins were visualized by incubation with a horseradish peroxidase (HRP)-conjugated anti-goat or rabbit IgG (1:2000; Santa Cruz Biotechnology) followed by development with a chemiluminescence substrate for HRP (Thermo Fisher Scientific). The images of western blots were captured by GE imageQuant imager. Relative band intensities were analyzed using Image J software and normalized to GAPDH.
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