Horseradish peroxidase conjugated anti rabbit and anti mouse secondary antibodies

Manufactured by Santa Cruz Biotechnology

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Horseradish peroxidase-conjugated anti-rabbit and anti-mouse secondary antibodies are lab equipment used in various immunoassay techniques. They are designed to bind to primary antibodies raised in rabbit or mouse, respectively, and the conjugated horseradish peroxidase enzyme can be used for detection and signal amplification.

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7 protocols using horseradish peroxidase conjugated anti rabbit and anti mouse secondary antibodies

Pharmacological Modulation of NR1, CaMKII, and CREB

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Morphine hydrochloride (First Pharmaceutical Factory of Shenyang, Shenyang, China) was dissolved in 0.9% saline to a final concentration of 1.0 mg/mL. ACPC and L-701,324 (Sigma-Aldrich, St. Louis, MO) were suspended in a 1% aqueous solution of Tween-80 to obtain concentrations of 20 mg/mL and 5 mg/mL, respectively. All drugs were injected i.p. at a volume of 10 mL/kg. Drug doses were chosen based on previous works (T. Li et al., 2010 (link); Y. P. Wang et al., 2015c (link)), pilot experiments, and other behavioral studies (Poleszak et al., 2007 (link); Labrie et al., 2008 (link); Skolnick et al., 2015 (link)). Rabbit polyclonal antibodies against phospho-NR1 (Ser890), phospho-CaMKII (Thr286), phospho-CREB (Ser133), and their total proteins were purchased from Cell Signaling Technology (Danvers, MA). Mouse monoclonal antibodies against GAPDH and the horseradish peroxidase-conjugated anti-rabbit and anti-mouse secondary antibodies were purchased from Santa Cruz Technology (Santa Cruz, CA).

Wang Y., Yin F., Guo H., Zhang J., Yan P, & Lai J. (2017). The Role of Dopamine D1 and D3 Receptors in N-Methyl-D-Aspartate (NMDA)/GlycineB Site-Regulated Complex Cognitive Behaviors following Repeated Morphine Administration. International Journal of Neuropsychopharmacology, 20(7), 562-574.

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BLM Purification and Cell Culture Assays

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BLM sulfate purified from Streptomyces verticillus was obtained from Sigma-Aldrich (St. Louis, MO, USA). Recombinant human TGF-β1 was purchased from R&D Systems (Minneapolis, MN, USA). Minimum essential medium (MEM), Dulbecco’s modified Eagle’s medium (DMEM)/Ham’s F12, fetal bovine serum (FBS), penicillin, and streptomycin were purchased from Life Technologies (Grand Island, NY, USA). Antibodies against Akt, phospho-Akt, Smad2/3, and phospho-Smad2/3 were obtained from Cell Signaling Technology (Danvers, MA, USA); antibodies against ASC, caspase-1, fibronectin, IL-1β, IL-18, NLRP3, SOD, and β-actin were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Antibodies raised against TGF-β1 and P2X ₇R were purchased from Abcam (Cambridge, MA, USA). Antibodies against α-SMA and collagen, type III, alpha 1 chain (collagen 3α1) were obtained, respectively, from Sigma-Aldrich and Acris Antibodies (San Diego, CA, USA). Horseradish peroxidase-conjugated anti-rabbit and anti-mouse secondary antibodies were obtained from Santa Cruz Biotechnology.

Huang T.T., Lai H.C., Ko Y.F., Ojcius D.M., Lan Y.W., Martel J., Young J.D, & Chong K.Y. (2015). Hirsutella sinensis mycelium attenuates bleomycin-induced pulmonary inflammation and fibrosis in vivo. Scientific Reports, 5, 15282.

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Western Blot Analysis of Cell Cycle Regulators

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Cells lysates were prepared with Pro-Prep lysis buffer (Intron Biotechnology, Seongnam, Korea). The protein samples were separated by SDS PAGE and transferred to polyvinylidene difluoride membranes. After blocking with 5% bovine serum albumin at room temperature for 1 hour, the membranes were incubated with primary antibodies at 4°C overnight, washed, and then incubated with the appropriate secondary antibody for 1 hour at room temperature. The signals were detected using an ECL Kit (Bio-Rad Laboratories, Hercules, CA, USA). The primary antibodies for p21, p27, and Skp2, and horseradish peroxidase-conjugated anti-rabbit and anti-mouse secondary antibodies, were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Antibodies against cyclin-dependent kinase 2 (CDK2), cyclin-dependent kinase 4 (CDK4), cyclin-dependent kinase 6 (CDK6), cyclin E1, cyclin D1, and tubulin were purchased from Cell Signaling Technology, Inc (Beverly, MA, USA).

Zhang H., Yi J., Yoon D., Ryoo Z., Lee I, & Kim M. (2020). Imatinib and GNF-5 Exhibit an Inhibitory Effect on Growth of Hepatocellar Carcinoma Cells by Downregulating S-phase Kinase-associated Protein 2. Journal of Cancer Prevention, 25(4), 252-257.

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Quantifying Bcl-2 Protein Expression

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To assay for changes in Bcl-2 protein levels, non-transfected and NP-siRNA complex-transfected cells were collected and lysed in RIPA lysis buffer. The cell lysate was collected by centrifugation. Total protein concentration was determined using a Bradford micro protein assay protocol (Sigma-Aldrich). Next, 30 μg of total protein from each sample were loaded on each well of 10% sodium dodecyl sulfate-polyacrylamide gel and electrophoresed. The proteins were transferred to polyvinylidene difluoride membranes and blocked with 5% non-fat dry milk (Santa Cruz Biotechnology) in Tris-buffered saline with Tween 20 for 1 hour. The blots were hybridized overnight at 4°C with primary monoclonal anti-Bcl-2 (Santa Cruz Biotechnology) and anti-β-actin (Abcam) antibodies, followed by incubation with horseradish peroxidase-conjugated anti-rabbit and anti-mouse secondary antibodies (Santa Cruz Biotechnology). Levels of proteins were detected using Crescendo chemiluminiscent detection reagents (Millipore, Billerica, MA, USA) and visualized using a UVP Biospectrum 810 imaging system (Ultra Violet Products Ltd, Cambridge, UK). Protein expression in each sample was quantified by densitometry using UVP software, normalized to β-actin levels, and then expressed relative to the non-transfected controls. Histograms were drawn.

Reddy T.L., Krishnarao P.S., Rao G.K., Bhimireddy E., Venkateswarlu P., Mohapatra D.K., Yadav J., Bhadra U, & Pal Bhadra M. (2015). Para amino benzoic acid-derived self-assembled biocompatible nanoparticles for efficient delivery of siRNA. International Journal of Nanomedicine, 10, 6411-6424.

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Antibody Characterization for Biochemical Analysis

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Rabbit polyclonal antibodies were: PKD, phospho-PKD-S⁹¹⁶ and phospho-PKD-S^744/748, p38 and phospho-p38-T¹⁸⁰/Y¹⁸², IKKβ, NF-κB phospho-p65-S⁵³⁶ (Cell Signaling Technology, Beverly, MA, USA); phospho-S^176/177 IKKα/β (Biorbyt, San Francisco, CA, USA); NSE (ICN Biomedicals; Costa Mesa, CA, USA); total and phospho-S³⁰⁸ DAPK (Sigma-Aldrich); Superoxide dismutase 2 (SOD2), microtubule-associated protein 2 (MAP2), PKD/PKC mu (phospho-Y463) (Abcam); GFP (ThermoFisher Scientific Invitrogen, Waltham, MA, USA); NF-κB p65, IκBα, DUSP1 (Santa Cruz Biotechnology, CA, USA). Mouse monoclonals were: NeuN, Spectrin (Millipore Corporation, Billerica, MA, USA), STEP (Novus Biologicals, Littleton, CO, USA), MDA (Jaica, Japan). Detailed information about all the above-mentioned antibodies and dilutions used for the different applications is given in Supplementary Table 2. Horseradish peroxidase-conjugated anti-rabbit and anti-mouse secondary antibodies were from Santa Cruz Biotechnology and Alexa-Fluor-488, -555, and -647 conjugated antibodies were from ThermoFisher Scientific.

Pose-Utrilla J., García-Guerra L., Del Puerto A., Martín A., Jurado-Arjona J., De León-Reyes N.S., Gamir-Morralla A., Sebastián-Serrano Á., García-Gallo M., Kremer L., Fielitz J., Ireson C., Pérez-Álvarez M.J., Ferrer I., Hernández F., Ávila J., Lasa M., Campanero M.R, & Iglesias T. (2017). Excitotoxic inactivation of constitutive oxidative stress detoxification pathway in neurons can be rescued by PKD1. Nature Communications, 8, 2275.

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Immunohistochemical Analysis of Grp58 and β-catenin in Cervical Cancer

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Formalin-fixed and paraffin-embedded tissues were examined using IHC, according to previously described procedures
[11 (link)]. The Grp58 (Atlas, 1:2000 dilution) and β-catenin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA; 1:100 dilution) antibodies were used, along with horseradish peroxidase-conjugated anti-rabbit and anti-mouse secondary antibodies (Santa Cruz). Immunocomplexes were visualized using the Envision kit (DAKO, Carpinteria, CA). Brown-colored cytoplasmic patches were considered Grp58-positive. Slides were scored separately by two independent pathologists (Y.L and S.M.J) blinded to all clinical data. Staining intensity was graded as absent (0), weak (1+), medium (2+) or strong (3+). The histoscore (Q) was calculated by multiplying the percentage (P) of positive cells by intensity (I), according to the formula: Q = P × I. The mean Q of each cervical cancer type was selected as the cut-off value to divide the high/low expression groups, as described previously.

Liao C.J., Wu T.I., Huang Y.H., Chang T.C., Lai C.H., Jung S.M., Hsueh C, & Lin K.H. (2014). Glucose-regulated protein 58 modulates β-catenin protein stability in a cervical adenocarcinoma cell line. BMC Cancer, 14, 555.

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Morphine Administration and BDNF Detection

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Morphine hydrochloride was purchased from Sigma Chemical Co. (St. Louis, MO, USA) and dissolved in sterile saline. The morphine dose used in the current study was 1.0 mg/mL. All injections were administered intraperitoneally (ip) in a volume of 10 mL/kg body weight. The primary antibodies against BDNF were from Abcam Technology (Cambridge, MA, USA). The primary antibodies for b-actin and the horseradish peroxidase-conjugated anti-rabbit and anti-mouse secondary antibodies were purchased from Santa Cruz Technology (Santa Cruz, CA, USA).

Wang Y.P., Wei S.G., Zhu Y.S., Zhao B., Xun X, & Lai J.H. (2015). Dopamine receptor D1 but not D3 essential for morphine-induced conditioned responses. Genetics and molecular research : GMR, 14(1).

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