Hrp conjugated secondary ab

Manufactured by Merck Group

Sourced in United States

The HRP conjugated secondary Ab is a horseradish peroxidase (HRP) labeled antibody used for detection and quantification in various immunoassay techniques. The HRP enzyme serves as a reporter molecule, catalyzing reactions that produce a measurable signal, enabling the visualization and quantification of target analytes.

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Secondary horseradish peroxidase (HRP)-conjugated Abs, (5 citations) Secondary Abs (3 citations) HRP-conjugated goat anti-rabbit secondary Abs (3 citations)

4 protocols using hrp conjugated secondary ab

Western Blot Protein Detection Protocol

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Immunoprecipitated complexes and cell lysates were resolved on either 16% Tris-Tricine SDS or 15% glycine SDS-PAGE and then transferred onto nitrocellulose membranes. The blots were probed with the appropriate combination of primary Ab and HRP conjugated secondary Ab (Sigma-Aldrich, USA). Recombinant His-tagged proteins were detected with rabbit anti-His Ab (Abcam Inc, USA), β2M with rabbit anti-β2M Ab (Abcam Inc, USA) and β-actin with mouse anti-β-actin Ab (Santa Cruz Biotechnology, USA). Bound HRP enzyme was detected by chemiluminescence following the manufacturer's protocol (GE Healthcare, UK).

Sreejit G., Ahmed A., Parveen N., Jha V., Valluri V.L., Ghosh S, & Mukhopadhyay S. (2014). The ESAT-6 Protein of Mycobacterium tuberculosis Interacts with Beta-2-Microglobulin (β2M) Affecting Antigen Presentation Function of Macrophage. PLoS Pathogens, 10(10), e1004446.

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Western Blot Analysis of Protein Extracts

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For all Western blot analyses, 1 × 10⁷ cells were collected, washed with PBS supplemented with protease inhibitor cocktail (11836153001, Sigma-Aldrich) and 1 mM PMSF (Sigma-Aldrich), and then lysed with Laemmli lysis buffer as previously described (46 (link)). Equal amounts of the protein extracts (~10 μg) were electrophoresed on NuPAGE 4–12% Bis-Tris gradient gels (Novex, Carlsbad, CA) and then electro-blotted onto Immobilon PVDF membranes (EMD Millipore, Billerica, MA). Membranes were probed with the following Abs and dilutions: GABPα (1:1000; SC-22810), PU.1 (1:1000; SC-352), C/EBPε (1:1000; SC-158), and β-actin (1:1000) (all from Santa Cruz Biotechnology, Dallas, TX), LBR from guinea pig serum [kindly provided by Prof. Harald Herrmann, German Cancer Research Institute, Heidelberg, Germany and previously described (20 (link))], or tubulin (1:1500; Sigma-Aldrich). HRP-conjugated secondary Ab (1:2000, Sigma-Aldrich) was used against primary Abs, and chemiluminescence was detected using Immobilon Western HRP substrate solution (EMD Millipore). Images were captured from exposed and processed film or with the ChemiDocMP Imager using Image Lab Software (BioRad Laboratories).

Malu K., Garhwal R., Pelletier M.G., Gotur D., Halene S., Zwerger M., Yang Z.F., Rosmarin A.G, & Gaines P. (2016). Cooperative activity of GABP with PU.1 or C/EBPε regulates lamin B receptor gene expression, implicating their roles in granulocyte nuclear maturation. Journal of immunology (Baltimore, Md. : 1950), 197(3), 910-922.

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Oleanolic Acid Modulates Glucose Metabolism

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Oleanolic acid (structure shown in Figure 1) was obtained from the Center for Drug Discovery at China Pharmaceutical University. Metformin (purity > 98%) was obtained from Sigma (St. Louis, MO, USA). The mouse insulin enzyme immunoassay ELISA kit was purchased from Invitrogen. Serum triglyceride (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) kits were purchased from Whitman Biotech (Nanjing, Ltd., China). Antibody (Ab) for G-6-Pase was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). TORC2 was from Millipore (Billerica, MA, USA), and phospho-CREB and β-actin were from Abcam (Cambridge, MA, USA). The Abs against protein kinase B (PKB/Akt), phospho-Ser473-Akt (pAkt), PI3K, phospho-PI3K, AMPK, phosphor-AMPK, ACC, phosphor-ACC, PEPCK1, mTOR, and phospho-mTOR were purchased from Cell Signaling Technology (Beverly, MA, USA). The HRP-conjugated secondary Abs, affinity-purified mouse anti-rabbit IgG, and rabbit anti-mouse IgG were purchased from Sigma (St. Louis, MO, USA).

Wang X., Chen Y., Abdelkader D., Hassan W., Sun H, & Liu J. (2015). Combination Therapy with Oleanolic Acid and Metformin as a Synergistic Treatment for Diabetes. Journal of Diabetes Research, 2015, 973287.

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Western Blot Analysis of Cell Signaling Proteins

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Western blotting was carried out as previously described.⁵ (link) Connexin32 Ab was purchased from Santa Cruz Biotechnology. The GAPDH Ab was obtained from Ray Antibody Biotech. β‐Tubulin Ab and HRP‐conjugated secondary Abs were acquired from Sigma‐Aldrich. The RIP1, RIP3, MLKL, p‐RIP1, p‐MLKL, and Src Abs were obtained from Cell Signaling Technology. Phosphor‐caspase 8 (Tyr380) primary Ab was from Abcam.

Xiang Y., Peng F., Guo Y., Ge H., Cai S., Fan L., Peng Y., Wen H., Wang Q, & Tao L. (2021). Connexin32 activates necroptosis through Src‐mediated inhibition of caspase 8 in hepatocellular carcinoma. Cancer Science, 112(9), 3507-3519.

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