Horseradish peroxidase conjugated mouse anti rabbit igg

Manufactured by Santa Cruz Biotechnology

Sourced in United States

Horseradish peroxidase-conjugated mouse anti-rabbit IgG is a secondary antibody used in immunoassays to detect and quantify rabbit primary antibodies. The horseradish peroxidase enzyme conjugated to the antibody can be used to catalyze a colorimetric or chemiluminescent reaction, allowing for the visualization and measurement of target proteins.

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Horseradish peroxidase (230 citations) IgG mouse (6 citations) Rabbit anti-IgG (4 citations) IgG Rabbit (3 citations) Mouse IgGs (3 citations) Horseradish peroxidase anti-rabbit (2 citations) Mouse anti-IgG (2 citations) Rabbit IgGs (2 citations) Rabbit anti-TM (2 citations) Horseradish peroxidase-conjugated anti-rabbit and anti-mouse IgGs (2 citations)

10 protocols using horseradish peroxidase conjugated mouse anti rabbit igg

Kidney Protein Quantification and Analysis

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Kidney lysates were prepared by homogenization on ice for 30 min using radioimmunoprecipitation assay buffer [50 mM Tris–Cl (pH 7.6), 150 mM NaCl, 1 % NP-40, 0.1 % sodium dodecyl sulfate, 0.5 % deoxycholic acid, 1 μg/mL leupeptin, 1 μg/mL aprotinin, and 0.5 mM phenylmethylsulfonyl fluoride], and then centrifuged at 12,000 rpm for 30 min at 4 °C, after which supernatants were collected. A total of 100 μg of protein was separated by 15 % sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. Anti-Bax, -Bcl-2, and -cleaved caspase-3 antibodies (Cell Signaling Technology, Danvers, MA, USA) were used as primary antibodies at 4 °C overnight, and horseradish peroxidase-conjugated anti-rabbit/mouse IgG (Santa Cruz Biotechnology) was used as the secondary antibody. Densitometry was performed using Quantity One software (Bio-Rad Laboratories, Hercules, CA, USA).

Luo C.J., Luo F., Zhang L., Xu Y., Cai G.Y., Fu B., Feng Z., Sun X.F, & Chen X.M. (2016). Knockout of interleukin-17A protects against sepsis-associated acute kidney injury. Annals of Intensive Care, 6, 56.

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Kidney Immune Protein Expression Analysis

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The RORγt, IL-17, Foxp3, and IL-10 protein levels were measured via western blotting (n = 5 per group). Total proteins were extracted from the kidney tissue using a complete radioimmunoprecipitation assay (RIPA) lysis buffer, and protein levels were measured using a bicinchoninic acid assay kit (Thermo Scientific, Bremen, Germany). A total of 100 μg of protein was separated and transferred onto polyvinylidene difluoride (PVDF) membranes. Then, the primary antibodies anti-RORγt, anti-IL-17, anti-Foxp3, anti-IL-10 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and anti-actin (Cell Signaling Technology) were incubated with the membranes at 4 °C overnight, and horseradish peroxidase-conjugated anti-rabbit/mouse IgG (Santa Cruz Biotechnology) was used as the secondary antibody. Protein levels were semiquantitatively determined based on the optical density using ImageJ software and are expressed as the protein/actin ratio. Sample quantification was performed twice.

Luo C., Luo F., Che L., Zhang H., Zhao L., Zhang W., Man X., Bu Q., Luan H., Zhou B., Zhou H, & Xu Y. (2023). Mesenchymal stem cells protect against sepsis-associated acute kidney injury by inducing Gal-9/Tim-3 to remodel immune homeostasis. Renal Failure, 45(1), 2187229.

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Western Blot Analysis of 4T1 Cells

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4T1 cells were lysed in a lysis buffer (Cell Signaling, Danvers, MA, USA), briefly sonicated, incubated on ice for 30 min and then centrifuged at 12,000× g for 10 min. The protein concentrations were measured by BCA protein assay. Ten µg of cell lysate was loaded on a precast polyacrylamide gel (Thermo-Fisher, Carlsbad, CA, USA), and the proteins were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Life Technologies) and transferred onto nitrocellulose membranes. After overnight incubation with a primary antibody, the membrane was washed and incubated with horseradish peroxidase-conjugated rabbit anti-mouse IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The presence of the protein of interest was visualized and quantitated with C-DiGit Blot scanner (Scrum, Tokyo, Japan).

Imamura M., Li T., Li C., Fujisawa M., Mukaida N., Matsukawa A, & Yoshimura T. (2021). Crosstalk between Cancer Cells and Fibroblasts for the Production of Monocyte Chemoattractant Protein-1 in the Murine 4T1 Breast Cancer. Current Issues in Molecular Biology, 43(3), 1726-1740.

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Quantification of Osteogenic Markers

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Proteins were extracted from the BMSCs from each group using RIPA buffer, according to the manufacturer’s instructions. A BCA Protein Assay kit (Beyotime, Beijing, China) was used to determine the protein concentrations. Equal amounts of proteins were loaded onto sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were incubated with the primary antibodies overnight at 4 °C (1:300 dilution, monoclonal mouse anti-PPARγ, anti-GREM1, anti-Runx2, anti-Bmp-2, or anti-C/EBPα; Santa Cruz Biotechnology, Dallas, TX, USA), followed by incubation with a secondary antibody (1:3,000 dilution, horseradish peroxidase-conjugated rabbit anti-mouse IgG, Santa Cruz Biotechnology, Santa Cruz, USA) at 37 °C for 1 h. The blots were examined using a chemiluminescence detection kit (Amersham Pharmacia Biotech, Piscataway, NJ, USA). An antibody against GAPDH (Santa Cruz Biotechnology, Santa Cruz, USA) served as an internal reference.

Gu C., Xu Y., Zhang S., Guan H., Song S., Wang X., Wang Y., Li Y, & Zhao G. (2016). miR-27a attenuates adipogenesis and promotes osteogenesis in steroid-induced rat BMSCs by targeting PPARγ and GREM1. Scientific Reports, 6, 38491.

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Western Blot Analysis of Osteogenic Markers

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Total proteins of cultured cells were extracted using RIPA buffer containing phenylmethanesulfonylfluoride. Protein concentration was determined using the BCA protein assay kit (Beyotime, Haimen, China) according to the manufacturer’s protocol. Proteins were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes. After blocking, the membranes were incubated overnight at 4°C with diluted (1:300) primary antibodies (monoclonal mouse anti-PPARγ or anti-C/EBPα or anti-OCN or anti-Runx2; Santa Cruz Biotechnology, Dallas, TEX, USA). Following extensive washing, the membranes were incubated with diluted (1:3000) horseradish peroxidase-conjugated rabbit anti-mouse IgG (Santa Cruz Biotechnology, Dallas, TEX, USA). Signals were determined using a chemiluminescence detection kit (Amersham Pharmacia Biotech, Piscataway, NJ, USA). An antibody against β-actin (Santa Cruz Biotechnology, Dallas, TEX, USA) served as endogenous reference.

Sun J., Wang Y., Li Y, & Zhao G. (2014). Downregulation of PPARγ by miR-548d-5p suppresses the adipogenic differentiation of human bone marrow mesenchymal stem cells and enhances their osteogenic potential. Journal of Translational Medicine, 12, 168.

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Immunohistochemical Detection of HMGA2

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Formalin-fixed and paraffin-embedded tissues were sectioned at 4 μm. After paraffin sections are routinely deparaffinized to water, antigen retrieval was performed by high temperature ethylene diamine tetraacetic acid (EDTA) solution. All immunohistochemical staining procedures were performed on a Leica ST5010 Autostainer XL (Buffalo Grove, USA). 1:50 mouse anti-HMGA2 monoclonal antibody and horseradish peroxidase-conjugated mouse-anti-rabbit IgG were used as primary and secondary antibodies (Santa Cruz Biotech., USA); DAB was used for color development with hematoxylin counterstaining. The negative control is PBS instead of the primary antibody. HMGA2 protein is positive in brownish-yellow.

Liu Y., Lv G., Bai J., Song L., Ding E., Liu L., Tian Y., Chen Q., Li K., Liu X, & Ding Y. (2022). Effects of HMGA2 on the epithelial-mesenchymal transition-related genes in ACHN renal cell carcinoma cells-derived xenografts in nude mice. BMC Cancer, 22, 421.

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Investigating Cellular Signaling Pathways

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Primary antibodies against p110δ, β-actin, Akt, and p-Akt were purchased from Cell Signaling Technology (Danvers, MA), and an antibody against dCasX was ordered from Hangzhou Huaan Biotechnology Co., Ltd (Hangzhou, Zhejiang, China). Alexa fluorescence-488-conjugated anti-CD11β antibody was purchased from Abcam (Cambridge, UK), Alexa fluorescence-532-conjugated anti-Panck antibody was from Nanostring (Nanostring Technologies, Seattle, WA), and Alexa fluorescence-594-conjugated mouse endothelial-specific IB4 was purchased from Life Technology (Grand Island, NY). Alexa fluorescence-488-conjugated mouse anti-rabbit IgG and Alexa fluorescence-594-conjugated mouse anti-rabbit IgG were ordered from Hangzhou Huaan Biotechnology Co., Ltd. Horseradish peroxidase-conjugated mouse anti-rabbit IgG and goat anti-mouse IgG were purchased from Santa Cruz Biotechnology (Dallas, TX). Enhanced chemiluminescent substrate to detect horseradish peroxidase was purchased from Thermo Scientific (Waltham, MA). The plasmid of pAAV-RSV-SpCas9 was from our own laboratory deposited to Addgene (Cat. 85450, Cambridge, MA).⁵² (link) High-fidelity Herculase II DNA polymerases were from Agilent Technologies (Santa Clara, CA).

Han H., Yang Y., Jiao Y., Qi H., Han Z., Wang L., Dong L., Tian J., Vanhaesebroeck B., Li X., Liu J., Ma G, & Lei H. (2023). Leverage of nuclease-deficient CasX for preventing pathological angiogenesis. Molecular Therapy. Nucleic Acids, 33, 738-748.

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Isolation and Characterization of Chalcomoracin

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As determined by HPLC/UV‐visible, 98.5% pure Chalocomoracin (CMR) was isolated by Jingkui Tian's laboratory, the Key Laboratory of Biomedical Engineering, Zhejiang University. The compounds were dissolved in pure DMSO and stored at −20°C prior to use. Primary antibodies against p‐Akt, Akt and p53 were purchased from Cell Signaling Technology, and β‐Actin was ordered from Santa Cruz Biotechnology. Horseradish peroxidase‐conjugated mouse anti‐rabbit IgG, and goat anti‐mouse IgG were ordered from Santa Cruz Biotechnology. Enhanced chemiluminescent substrate to detect horseradish peroxidase was purchased from Thermo Scientific.
ARPE‐19 cells (American Type Culture Collection) was cultured in Dulbecco's modified Eagle's medium/nutrient mixture (DMEM/F‐12, Invitrogen) supplemented with 10% foetal bovine serum (FBS). Cells were cultured in a humidified incubator at 37°C and 5% CO₂.
Detailed protocols of Western blot, cell proliferation assay, cell migration assay and collagen contraction assay were descripted in our previous report in.³²

Han H., Yang Y., Liu B., Tian J., Dong L., Qi H., Zhu W., Wang J, & Lei H. (2021). Chalcomoracin prevents vitreous‐induced activation of AKT and migration of retinal pigment epithelial cells. Journal of Cellular and Molecular Medicine, 25(19), 9102-9111.

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Western Blot Analysis of DNA Repair Proteins

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Whole cell extracts from DM-CE isolated from at least 3 mice per time-point were taken as described in our earlier publication (Liu et al., 2020 (link); Miyajima et al., 2020 (link)). UVA-irradiated CE tissue from one mouse was used per replicate for western blotting. Briefly, proteins were loaded into 4–12% Bis-Tris NuPAGE gels (#NP0336, Thermo Fisher Scientific, USA), run, and transferred to polyvinylidene difluoride membrane (PVDF), blocked with 5% non-fat dry milk, probed with primary antibodies overnight then exposed next day to secondary antibodies for 1-hour and finally probed with Super Signal West Pico or Femto (#34577 or #34096, Thermo Fisher Scientific, USA). Chemiluminescent signal was captured by exposing the blots to an x-ray film and developing it using an automated film processor inside a dark room. Densitometry was performed using Image J software (NIH, USA). The primary antibodies used were LIG3 (1:1000, 611876, BD Biosciences, USA), Neil2 (1:1000, GTX132565, Genetex), Top3a (1:1000, PA5-116710, Life Technologies), β-Actin (1:5000, A1978, Sigma, USA), and gapdh (1:2000, G9545, Sigma, USA) and secondary antibodies were horseradish peroxidase-conjugated mouse anti-rabbit IgG (1:1000, sc-2357) and anti-mouse IgG (1:1000, sc-516102) from Santa Cruz Biotechnology Inc., USA.

Ashraf S., Deshpande N., Vasanth S., Melangath G., Wong R.J., Zhao Y., Price M., Price F J.r, & Jurkunas U.V. (2023). Dysregulation of DNA repair genes in Fuchs Endothelial Corneal Dystrophy. Experimental eye research, 231, 109499.

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SARS-CoV-2 Spike Protein Detection

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Transfected HEK293T cells were lysed in cell lysis buffer (Cell Signaling Technology) and fractionated by SDS-PAGE (Any kD acrylamide gel, Bio-Rad). Proteins were detected by western blotting using anti-SARS-CoV-2 (2019-nCoV) Spike RBD, rabbit polyclonal antibody (1:2000 dilution; Sino Biological) and horseradish peroxidase-conjugated mouse anti-rabbit IgG (1:4000; Santa Cruz Biotechnology). The protein bands were visualized by enhanced chemiluminescence using ChemiDoc^TM XRS⁺ (Bio-Rad) and an Image Lab^TM Software (Bio-Rad).

Komori M., Nogimori T., Morey A.L., Sekida T., Ishimoto K., Hassett M.R., Masuta Y., Ode H., Tamura T., Suzuki R., Alexander J., Kido Y., Matsuda K., Fukuhara T., Iwatani Y., Yamamoto T., Smith J.F, & Akahata W. (2023). saRNA vaccine expressing membrane-anchored RBD elicits broad and durable immunity against SARS-CoV-2 variants of concern. Nature Communications, 14, 2810.

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