Horseradish peroxidase coupled secondary antibodies

Manufactured by Promega

Horseradish peroxidase-coupled secondary antibodies are a type of enzyme-linked immunosorbent assay (ELISA) reagent. They function as detection agents, binding to primary antibodies and catalyzing a colorimetric reaction to facilitate the visualization and quantification of target analytes.

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Lab products found in correlation

Gapdh, by Cell Signaling Technology (4 mentions) Chemidoc system, by Bio-Rad (3 mentions) Phosphorylated rbm20 s637, by Abmart (2 mentions) Supersignal west femto chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions) Ab5603, by Merck Group (1 mentions) Mab1378, by Merck Group (1 mentions) Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions) Integrin β1, by Merck Group (1 mentions) Mab1959, by Merck Group (1 mentions) Imagequant las 500 system, by GE Healthcare (1 mentions) Mab1729, by R&D Systems (1 mentions) Complete protease inhibitor cocktail, by Roche (1 mentions) Anti gapdh 14c10, by Cell Signaling Technology (1 mentions) Proteinase inhibitor, by Roche (1 mentions) Precision plus protein standard, by Bio-Rad (1 mentions) Fuji medical x ray film, by Fujifilm (1 mentions) Nitrocellulose membrane, by Bio-Rad (1 mentions) Dnm1l, by Santa Cruz Biotechnology (1 mentions) Pierce enhanced chemiluminescence, by Thermo Fisher Scientific (1 mentions) Mini protean tgx, by Bio-Rad (1 mentions)

Spelling variants of this product

Horseradish peroxidase (16 citations) Peroxidase-coupled secondary antibodies (4 citations)

6 protocols using horseradish peroxidase coupled secondary antibodies

Western Blotting Analysis of RBM20

Cited 2 times

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Western blotting analysis was performed as described previously.^{29 (link),30 (link)} Briefly, protein lysed from cells and tissues was separated by SDS-PAGE gel and transferred onto a PVDF membrane. The membrane was probed with primary antibodies against RBM20 (1:1500, rabbit, homemade^{3 (link)}), phosphorylated RBM20 S637 (1:1000, rabbit; Abmart Inc. Shanghai, China), GAPDH (1:1500, rabbit; Cell Signaling Technology, Danvers, Massachusetts, USA; Cat#2118S) served as the protein-loading control. Then, the membrane was probed with horseradish peroxidase-coupled secondary antibodies (1:3000, anti-rabbit; Promega; Cat#W4011) for 1 h. Chemiluminescence images were taken by ChemiDoc system (Bio-Rad, Hercules, CA).

Zhang Y., Wang C., Sun M., Jin Y., Braz C.U., Khatib H., Hacker T.A., Liss M., Gotthardt M., Granzier H., Ge Y, & Guo W. (2022). RBM20 phosphorylation and its role in nucleocytoplasmic transport and cardiac pathogenesis. The FASEB Journal, 36(5), e22302.

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Western Blot Analysis of Pluripotency Markers

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The following antibodies were used: OCT4 (SC8629, Santa Cruz Biotechnology), NANOG (MABD24, Millipore), SOX2 (AB5603, Millipore), Integrin α6 (MAB1378, Millipore), Integrin β1 (MAB1959, Millipore), PSMD11 (NBP2-59484, Novus Biologicals; Centennial, CO) and GAPDH (2118, Cell Signaling Technology). Whole-cell lysates were prepared from cells, separated on 10% SDS-polyacrylamide gel, and transferred to polyvinylidene difluoride membranes. The membranes were incubated with 5% milk in TBST (w/v) for 1 h and then incubated with primary antibodies diluted in 5% BSA in TBST overnight at 4 °C. Blots were incubated with horseradish peroxidase-coupled secondary antibodies (Promega, Madison, WI; R&D systems, Mckinley NE, MN) for 1 h, and protein expression was detected using SuperSignal West Pico Chemiluminescent Substrate or SuperSignal West Femto Chemiluminescent Substrate (Thermo Scientific, Waltham, MA). NIH ImageJ software was used for the quantification of blotting images. Uncropped and unprocessed scans for blots are in Supplementary Fig. 11. All blots were derived from the same experimental replicate and processed in parallel.

Timilsina S., Kirsch-Mangu T., Werth S., Shepard B., Ma T, & Villa-Diaz L.G. (2022). Enhanced self-renewal of human pluripotent stem cells by simulated microgravity. NPJ Microgravity, 8, 22.

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Western Blot Analysis of RBM20

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Western blotting analysis was performed as described previously.²⁹, ³⁰ Briefly, protein lysed from cells and tissues was separated by SDS‐PAGE gel and transferred onto a PVDF membrane. The membrane was probed with primary antibodies against RBM20 (1:1500, rabbit, homemade³), phosphorylated RBM20 S637 (1:1000, rabbit; Abmart Inc. Shanghai, China), GAPDH (1:1500, rabbit; Cell Signaling Technology, Danvers, Massachusetts, USA; Cat#2118S) served as the protein‐loading control. Then, the membrane was probed with horseradish peroxidase‐coupled secondary antibodies (1:3000, anti‐rabbit; Promega; Cat#W4011) for 1 h. Chemiluminescence images were taken by ChemiDoc system (Bio‐Rad, Hercules, CA).

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Western Blot Analysis of Liver and Brain Samples

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Fresh-frozen liver or brain samples (approximately 100 mg) were lysed in 5× volume of lysis buffer (150 mM NaCl, 10% glycerol, 2 mM EDTA, 0.5% NP-40, 0.1% sodium deoxycholate, 20 mM Hepes, 1× cOmplete Protease Inhibitor Cocktail [Roche], pH 7.4) using lysing-matrix D at 6,000 rpm for 40 s. The lysates were diluted 1:5 in lysis buffer and prepared for SDS-PAGE (10% or 15%). Primary antibodies used were 22C11 (Millipore), anti-GAPDH (14C10; Cell Signaling), TP2 (kind gift of the Ottawa Heart Institute), anti-triggering receptor expressed in myeloid cells 2 (TREM2) (Mab1729; R&D Systems) and anti-ABCA7 (polyclonal; Thermo Fisher Scientific), and horseradish peroxidase-coupled secondary antibodies (Promega). Chemiluminescence images were acquired using the ImageQuant LAS 500 system (GE Healthcare).

Oestereich F., Yousefpour N., Yang E., Phénix J., Nezhad Z.S., Nitu A., Vázquez Cobá A., Ribeiro-da-Silva A., Chaurand P, & Munter L.M. (2022). The cholesteryl ester transfer protein (CETP) raises cholesterol levels in the brain. Journal of Lipid Research, 63(9), 100260.

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Western Blot Analysis of Mitochondrial Proteins

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Whole-cell extracts were prepared using RIPA buffer containing proteinase inhibitors (Roche) and resolved using SDS-PAGE (Mini-PROTEAN TGX, Bio-Rad) under reducing conditions (β-mercaptoethanol, Bio-Rad) and Precision Plus Protein Standards (dual color, Bio-Rad) was used. Proteins were subsequently blotted onto a nitrocellulose membrane (Bio-Rad) and hybridized using anti-human antibodies against β-ACTIN (Sigma, 1:15,000), DNM1L (Santa Cruz Biotechnology, 1:200), MFN1 and MFN2 (Abcam, 1:1,000), PPARGC1A (Calbiochem, 1:1,000), and horseradish peroxidase–coupled secondary antibodies (1:6,000, Promega). The Pierce enhanced chemiluminescence (Thermo Fisher Scientific) detection system and Fuji Medical X-ray Films (FUJIFILM) was used. Quantification of densitometric units was performed using Scion Image (SCR_008673).

Nóbrega-Pereira S., Santos F., Oliveira Santos M., Serafim T.L., Lopes A.P., Coutinho D., Carvalho F.S., Domingues R.M., Domingues P., Bernardes de Jesus B., Morais V.A, & Dias S. (2023). Mitochondrial Metabolism Drives Low-density Lipoprotein-induced Breast Cancer Cell Migration. Cancer Research Communications, 3(4), 709-724.

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Protein Extraction and Western Blot Analysis

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Total proteins were lysed from heart LV tissues as described previously [8 ]. Briefly, proteins were separated by SDS-PAGE gel and transferred onto a PVDF membrane. The membrane was probed with primary antibodies against RBM20 (1:1000, rabbit), GAPDH (1:1500, rabbit; Cell Signaling Technology, Danvers, Massachusetts, USA; Cat#2118S) was served as the protein loading control. Then the membrane was probed with horseradish peroxidase-coupled secondary antibodies (1:3000, anti-rabbit; Promega; Cat#W4011) for 1 hour. Chemiluminescence images were taken by ChemiDoc system (Bio-Rad, Hercules, CA).
Subcellular proteins were extracted from LV at two-months-old mice followed the instruction (Thermo Fisher Scientific, Waltham, MA; Cat# 87790). Cytoplasmic extract and nuclear extract were loaded into SDS-PAGE gel and transferred onto PVDF membrane. The membrane was probed with primary antibodies against RBM20, HSP90 (1:1000, rabbit; Cell Signaling Technology, Danvers, Massachusetts, USA; Cat# #4874), GAPDH and Histone H3 (1:1500, rabbit; Cell Signaling Technology, Danvers, Massachusetts, USA; Cat# #4499). Incubation with secondary antibody and taken chemiluminescence images were descripted above.

Wang C., Zhang Y., Methawasin M., Braz C.U., Gao-Hu J., Yang B., Strom J., Gohlke J., Hacker T., Khatib H., Granzier H, & Guo W. (2022). RBM20S639G mutation is a high genetic risk factor for premature death through RNA-protein condensates. Journal of molecular and cellular cardiology, 165, 115-129.

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