Hrp conjugated mouse anti rabbit

Manufactured by Santa Cruz Biotechnology

The HRP-conjugated mouse anti-rabbit is a secondary antibody conjugated with Horseradish Peroxidase (HRP). This product is designed for use in various immunoassay techniques, such as Western blotting, ELISA, and immunohistochemistry, to detect and visualize target proteins that have been labeled with a primary rabbit antibody.

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3 protocols using hrp conjugated mouse anti rabbit

Immunoblot Analysis of Signaling Proteins

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Cells were lysed on ice in RIPA buffer with a protease and phosphatase inhibitor mixture (MilliporeSigma, P8340 & P5726), and immunoblots were performed as previously described (9 (link)). Primary antibodies used in this study were CYB5R3 (1:1,000; Proteintech, 10894-1-AP), β-actin (1:1,000; Cell Signaling Technology, 5125), Smad 2/3 (1:1,000; Cell Signaling Technology, 8685), p-Smad 2/3 (1:1,000; Cell Signaling Technology, 8828), ERK1/2 (1:1,000; Cell Signaling Technology, 4695), p-ERK1/2 (1:1,000; Cell Signaling Technology, 4370), caspase-3 (1:1,000; Cell Signaling Technology, 14220), cleaved caspase-3 (1:1,000; Cell Signaling Technology, 9661), and vimentin (1:1,000; Cell Signaling Technology, 5741). Detection and housekeeping antibodies used in this study are listed here: HRP-conjugated β-actin (1:30,000; Proteintech, HRP-66009), HRP-conjugated mouse anti-goat (1:2,000; Santa Cruz Biotechnology, sc-2354), and HRP-conjugated mouse anti-rabbit (1:2,000; Santa Cruz Biotechnology, sc-2357).

Bueno M., Calyeca J., Khaliullin T., Miller M.P., Alvarez D., Rosas L., Brands J., Baker C., Nasser A., Shulkowski S., Mathien A., Uzoukwu N., Sembrat J., Mays B.G., Fiedler K., Hahn S.A., Salvatore S.R., Schopfer F.J., Rojas M., Sandner P., Straub A.C, & Mora A.L. (2023). CYB5R3 in type II alveolar epithelial cells protects against lung fibrosis by suppressing TGF-β1 signaling. JCI Insight, 8(5), e161487.

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Quantitative Analysis of Protein Expression

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The frozen kidney of each rat was thawed, dissected into the cortex and medulla, and then homogenized in 100 mmol·L⁻¹ potassium buffer (pH 7.25) containing 30% glycerol, 1 mmol·L⁻¹ dithiothreitol, and 0.1 mmol·L⁻¹ phenylmethylsulfonyl fluoride (15 (link)). Protein expression and phosphorylation were examined using Western blot analysis, as described previously (19 (link)). Antibodies against Raf-B (no. 5284, Santa Cruz), ERK (no. 4695, Cell Signaling Technology), p-ERK (no. 4376, Cell Signaling Technology), mTOR (no. 2983, Cell Signaling Technology), p-mTOR (no. 2971, Cell Signaling Technology), S6 (no. 2217, Cell Signaling Technology), and p-S6 (no. 2211, Cell Signaling Technology) were used. Secondary HRP-conjugated mouse antirabbit (no. 2357, Santa Cruz) and rabbit antimouse (no. 516102, Santa Cruz) antibodies were then used. Relative band intensities were quantified using ImageJ and normalized using β-actin (A2228; Sigma-Aldrich, St. Louis, MO) as an internal standard.

QIU J., SATO Y., XU L., MIURA T., KOHZUKI M, & ITO O. (2021). Chronic Exercise Protects against the Progression of Renal Cyst Growth and Dysfunction in Rats with Polycystic Kidney Disease. Medicine and Science in Sports and Exercise, 53(12), 2485-2494.

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Kidney Protein Extraction and Analysis

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The frozen kidney of each rat was thawed, dissected into the cortex and medulla, and then homogenized in 100 mmol/L potassium buffer (pH 7.25) containing 30% glycerol, 1 mmol/L (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
dithiothreitol, and 0.1 mmol/L phenylmethylsulfonyl fluoride (14). Protein expression and phosphorylation were examined using western blot analysis, as described previously (18) .
Antibodies against Raf-B (#5284; Santa Cruz), ERK (#4695; Cell Signaling Technology), p-ERK (#4376; Cell Signaling Technology), mTOR (#2983; Cell Signaling Technology), p-mTOR (#2971; Cell Signaling Technology), S6 (#2217; Cell Signaling Technology), and p-S6 (#2211; Cell Signaling Technology) were used. Secondary HRP-conjugated mouse anti-rabbit (#2357; Santa Cruz) and rabbit anti-mouse (#516102; Santa Cruz) antibodies were then used. Relative band intensities were quantified using ImageJ and normalized using β-actin (A2228; Sigma-Aldrich, St.
Louis, MO, USA) as an internal standard.

Qiu J., Sato Y., Xu L., Miura T., Kohzuki M., & Ito O. (2021). Chronic exercise protects against the progression of renal cyst growth and dysfunction in rats with polycystic kidney disease.

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