Horseradish peroxidase hrp labeled secondary antibody

Manufactured by Merck Group

Sourced in United States

Horseradish peroxidase (HRP)-labeled secondary antibody is a type of laboratory reagent used in various immunoassay techniques. It consists of a secondary antibody that has been conjugated with the enzyme horseradish peroxidase. This enzyme can catalyze a colorimetric reaction, allowing for the detection and quantification of the target analyte in the sample.

Automatically generated - may contain errors

Lab products found in correlation

Close spelling variants of this product

Horseradish peroxidase (696 citations) Horseradish peroxidase (HRP) (213 citations) Horseradish peroxidase-labeled secondary antibody (17 citations) HRP-labeled secondary antibodies (12 citations) Peroxidase-labeled secondary antibodies (6 citations) Horseradish peroxidase (HRP)-labeled goat anti-mouse secondary antibody (2 citations)

10 protocols using horseradish peroxidase hrp labeled secondary antibody

Protein Expression Analysis of Cancer Markers

Check if the same lab product or an alternative is used in the 5 most similar protocols

Cells (3 × 10⁵) were seeded into 6-well plates and incubated for 24 h, lysed, centrifuged at 4℃ at 14,000 × g for 5 min. The concentration of protein in the supernatant was measured by a Bradford assay. A 100 μg protein sample was separated on a 12% SDS-PAGE gel and transferred onto a PVDF membrane. The membrane was blocked with 5% skim milk for 2 h at room temperature. Monoclonal antibodies against MDR1 (1:800, Santa Cruz), MRP1 (1:800, Santa Cruz), GST-π (1:800, Santa Cruz), CDX2 (1:800, Santa Cruz), Bcl-2 (1:800, Santa Cruz), Survivin (1:800, Santa Cruz), Topo-Ⅱ(1:800, Santa Cruz), p-AKT (1:500, Santa Cruz) and β-actin (1:5000, Sigma) were added and incubated at 4°C overnight in 5% BSA. The membrane was washed 3 times with Tris-buffered saline (TBS) containing 0.1% Tween 20. Horseradish peroxidase (HRP)-labeled secondary antibody (1:1000, Sigma) was added and incubated at room temperature for 1 h. After the last membrane wash, protein was visualized using a DAB kit (Millipore).

Wang Q., Wang Z., Chu L., Li X., Kan P., Xin X., Zhu Y, & Yang P. (2015). The Effects and Molecular Mechanisms of MiR-106a in Multidrug Resistance Reversal in Human Glioma U87/DDP and U251/G Cell Lines. PLoS ONE, 10(5), e0125473.

+ Open protocol

+ Expand

Protein Expression Analysis in Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

According to the manufacturer's instructions, the total protein in the cells or tissue was extracted using the RIPA buffer. 30ug of total proteins from each sample were separated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to the polyvinylidene fluoride membrane through a wet transfer system. After blocking the membranes, they were incubated with the primary antibodies against Bcl-2, Bax, TNF-α, Nrf2, Keap1, HO-1, GSH-Px, γ-GCS and NQO1 (all 1:800, Santa Cruz, USA). β-actin (1:5000, Sigma, USA) served as a loading control. Horseradish peroxidase (HRP)-labeled secondary antibody (1:1000, Sigma, USA) was used and incubated for 1 h at 25°C. The band densities were quantified by the LICOR Odyssey infrared imaging system (LICOR Bio- science, Nebraska, USA).

Ke Y., Yu K., Zeng W, & Lian G. (2019). Protective roles of Pyracantha fortuneana extract on acute renal toxicity induced by cadmium chloride in rats. Acta Cirúrgica Brasileira, 34(7), e201900706.

+ Open protocol

+ Expand

Western Blot Analysis of CDH1 Expression

Check if the same lab product or an alternative is used in the 5 most similar protocols

The treated A2780 and OVCAR3 cells were harvested, and proteins were extracted by RIPA (Beyotime, Shanghai, China). The concentration was measured by a Bicinchoninic acid (BCA) assay kit (Beyotime). Proteins were then fractionated and transferred to polyvinylidene difluoride (PVDF) membranes (GE Healthcare, Piscataway, NJ, USA). Next, the membranes were soaked in 5% milk powder for 2 h, and incubated with primary antibodies against CDH1 (1:1000, Cell Signaling Technology, Shanghai, China) or β-actin (1:2000, Abcam, Cambridge, MA, USA) overnight at 4 ℃. The membranes were incubated with horseradish peroxidase (HRP)-labeled secondary antibody (1:2000, Sigma-Aldrich) for 1 h. The bands were visualized using an ECL-PLUS kit (GE Healthcare, Piscataway, NJ, USA).

Lin C., Xu X., Yang Q., Liang L, & Qiao S. (2020). Circular RNA ITCH suppresses proliferation, invasion, and glycolysis of ovarian cancer cells by up-regulating CDH1 via sponging miR-106a. Cancer Cell International, 20, 336.

+ Open protocol

+ Expand

Western Blot Analysis of Hepatic Transcription Factors

Check if the same lab product or an alternative is used in the 5 most similar protocols

Cells were lysed with the RIPA lysis buffer. The cell lysate was subject to 10% SDS-polyacrylamide gel electrophoresis and proteins were transferred onto polyvinylidene difluoride (PVDF) membrane (Roche Diagnostics). The membrane was blocked with 5% blotting milk in PBST (20 mM Tris-HCl pH7.6, 137 mM NaCl, 0.5% Tween) and then incubated with one of the primary antibodies [anti-HNF1α (sc-6548, Santa Cruz), anti-HNF4α (C11F12, Cell Signaling Technology), anti-p65 (sc-372, Santa Cruz), anti-HDAC2 (sc-5549, Santa Cruz), anti-preS1 (sc57761, Santa Cruz), anti-flag (Sigma), and anti-β-actin (Sigma)] overnight at 4°C. The membrane was washed with PBST and incubated with the horseradish peroxidase (HRP)-labeled secondary antibody (Sigma) at room temperature for 2 hours. Signals were visualized with enhanced chemiluminescence detection reagents (Amersham).

Lin J., Gu C., Shen Z., Liu Y., Wang W., Tao S., Cui X., Liu J, & Xie Y. (2017). Hepatocyte nuclear factor 1α downregulates HBV gene expression and replication by activating the NF-κB signaling pathway. PLoS ONE, 12(3), e0174017.

+ Open protocol

+ Expand

Western Blot Analysis of PI3K/AKT Pathway

Check if the same lab product or an alternative is used in the 5 most similar protocols

After transfection for 24 hours, Hs 766T or SW1990 cells were lysed by Western cell lysis buffer (Beyotime, Haimen, People's Republic of China). An equal amount of protein samples was loaded onto sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel and blotted onto polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA). The membrane was incubated with 5% skimmed milk for 1 hour at room temperature for blocking. Primary antibodies against p-AKT (07–1398; Sigma), AKT (SAB4500797; Sigma), p-PI3K (ab389562; Abcam, Cambridge, MA, USA), PI3K (ab32089; Abcam), SLC7A11 (SAB2500951; Sigma) and β-actin (A1978; Abcam) were incubated with the membrane at 4°C overnight. Afterwards, horseradish-peroxidase (HRP)-labeled secondary antibody (Sigma) was incubated with the membrane for 2 hours at room temperature. Immunochemical detection was conducted with the enhanced chemiluminescence (ECL) Detection System (GE Healthcare, Chicago, IL, USA).

Zhang T., Li M., Lu H, & Peng T. (2021). Up-Regulation of circEIF6 Contributes to Pancreatic Cancer Development Through Targeting miR-557/SLC7A11/PI3K/AKT Signaling. Cancer Management and Research, 13, 247-258.

+ Open protocol

+ Expand

Protein Expression Analysis of Compound 7g in Colorectal Cancer

Check if the same lab product or an alternative is used in the 5 most similar protocols

Protein levels were analyzed by western blotting. Human colorectal cancer cell lines SW620 and HT29 treated with compound 7g for 48 h were collected and lysed in the lysis buffer containing RIPA (Radioimmunoprecipitation, Beyotime, Shanghai, China), a protease inhibitor and a phosphatase inhibitor (Roche, Mannheim, Germany). The concentration of proteins was measured by a BCA (Bicinchoninic acid) kit (Beyotime, P0010, Shanghai, China). Cell lysates (50 μg) were separated by 8–15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore Corporation, MA, USA). The membranes were blocked in TBST containing 5% BSA (Beyotime, ST023, Shanghai, China) for 2 h at room temperature and then incubated with primary antibodies (diluted by product specification, CST, USA) overnight at 4 °C. After that, the membranes were washed five times with TBST and then incubated with horseradish peroxidase (HRP)-labeled secondary antibodies (diluted 1:10000, Sigma, USA) for 1 h at room temperature. Subsequently, membranes were washed five times with TBST and then visualized with the ECL western blot detection kit (GE Healthcare, RPN3244, USA) on a Tanon 5200 Imaging System (Tanon Science & Technology Co., Ltd, Shanghai, China).

He L.J., Yang D.L., Li S.Q., Zhang Y.J., Tang Y., Lei J., Frett B., Lin H.K., Li H.Y., Chen Z.Z, & Xu Z.G. (2018). Facile construction of fused benzimidazole-isoquinolinones that induce cell-cycle arrest and apoptosis in colorectal cancer cells. Bioorganic & medicinal chemistry, 26(14), 3899-3908.

+ Open protocol

+ Expand

Dopamine receptor signaling study in HEK293 cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

Human embryonic kidney cells (HEK-293) were obtained from the American Type Culture Collection (Rockville, MD, USA). Cell culture media and fetal bovine serum were obtained from Life Technologies, Inc. (Carlsbad, CA, USA). Dopamine (DA), (-) quinpirole, haloperidol, PMA, anti-Flag M2 antibodies, anti-Flag-conjugated agarose beads, antibodies for green fluorescence protein (GFP), horseradish peroxidase (HRP)-labeled secondary antibodies, and glutathione beads were purchased from Sigma/Aldrich Chemical (St Louis, MO, USA). [³H]-Sulpride (87 Ci/mmol) and [³H]-spiperone (85.7 Ci/mmol) were purchased from PerkinElmer Life Sciences (Boston, MA, USA).

Zheng M., Zhang X., Min C., Choi B.G., Oh I.J, & Kim K.M. (2016). Functional Regulation of Dopamine D3 Receptor through Interaction with PICK1. Biomolecules & Therapeutics, 24(5), 475-481.

+ Open protocol

+ Expand

Quantitative Western Blot Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols

Whole-cell lysates were prepared by resuspending cell pellets in lysis buffer [20 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4, 1 μg/mL leupeptin, 1 μg/mL aprotinin, 1 μM PMSF] in culture flasks. Protein concentrations were measured using DC Protein Assay kit (Bio-Rad, Hercules, CA). Equal amount of proteins (30 μg) were separated with 10% SDS–polyacrylamide gel and transferred on to a PVDF membrane (Amersham, Piscataway, NJ). Membranes were blocked with 5% w/v non-fat dry milk dissolved in Tris buffered saline with Tween-20 (TBS-T; 0.1% Tween-20; pH 8.3) at room temperature for 1 h, then incubated with primary antibodies at 4 °C overnight. The primary antibodies used were rabbit COX-2 or β-actin antibodies (Santa Cruz, CA). After washing with TBS-T, membranes were incubated with horseradish peroxidase (HRP)-labeled secondary antibodies (Sigma, USA) for 30–45 min at room temperature. Immunobands were visualized using enhanced chemiluminescence (ECL) kit (Amersham). The amounts of each protein was quantified relative to that of β-actin by densitometry.

Chen Y., Li L.B., Zhang J., Tang D.P., Wei J.J, & Zhuang Z.H. (2018). Simvastatin, but not pravastatin, inhibits the proliferation of esophageal adenocarcinoma and squamous cell carcinoma cells: a cell-molecular study. Lipids in Health and Disease, 17, 290.

+ Open protocol

+ Expand

Western Blot Analysis of Protein Expression

Check if the same lab product or an alternative is used in the 5 most similar protocols

Total protein was extracted from cells using lysis buffer containing 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM EDTA, 1% Triton-X 100, 1% DTT, and 1% protease inhibitor cocktail (Roche, Basel, Switzerland). Equal amounts of protein extracts (40 μg) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a PVDF membrane. Membranes were blocked with 5% w/v non-fat dry milk dissolved in Tris buffered saline plus Tween-20 (TBS-T; 0.1% Tween-20; pH 8.3) at room temperature for 1 h, then incubated with primary antibodies at 4 °C overnight. The following antibodies were used for Western blotting: mouse monoclonal anti-β-actin (A5441, Sigma-Aldrich, MO, USA), rabbit polyclonal anti-CREB3L1 (ab33051, Abcam, MA, USA), and rabbit polyclonal anti-FGFBP1 (sc-292235, Santa Cruz Biotechnology, TX, USA). After washing with TBS-T, membranes were incubated with horseradish peroxidase (HRP)-labeled secondary antibodies (Sigma, USA) for 1 h at room temperature. Immunobands were visualized using enhanced chemiluminescence (ECL) kit (GE Healthcare, Waukesha, WI, USA) according to manufacture’s instructions.

Zhu H.Y., Bai W.D., Liu J.Q., Zheng Z., Guan H., Zhou Q., Su L.L., Xie S.T., Wang Y.C., Li J., Li N., Zhang Y.J., Wang H.T, & Hu D.H. (2016). Up-regulation of FGFBP1 signaling contributes to miR-146a-induced angiogenesis in human umbilical vein endothelial cells. Scientific Reports, 6, 25272.

+ Open protocol

+ Expand

Antibody Procurement and Preparation

Check if the same lab product or an alternative is used in the 5 most similar protocols

Anti-RPL13, anti-DDX3, and anti-G3BP1 monoclonal antibodies were purchased from Abcam (Cambridge, MA, USA). Anti-green fluorescent protein (GFP), anti-FLAG, and anti-β-actin monoclonal antibodies were purchased from Santa Cruz Biotechnology (CA, USA). Anti-hemagglutinin (HA) monoclonal antibody was purchased from Cell Signaling Technology (Danvers, MA, USA). Horseradish peroxidase (HRP)-labeled secondary antibodies were purchased from Sigma-Aldrich (St. Louis, MO, USA). Polyclonal pig antiserum directed against FMDV was prepared and stored in our laboratory. Lipofectamine LTX Reagent for the transfection of recombinant plasmids and Lipofectamine RNAiMax Reagent for the transfection of siRNA were purchased from Invitrogen (CA, USA). The QuikChange Site-Directed Mutagenesis Kit was purchased from Agilent Technologies (CA, USA). RNA was extracted with TRIzol Reagent, purchased from Invitrogen (CA, United States). The specific inhibitors of helicase DDX3, RK-33 and ketorolac salt, were purchased from Selleck Chemicals (Houston, TX, USA).

Guan J., Han S., Wu J., Zhang Y., Bai M., Abdullah S.W., Sun S, & Guo H. (2021). Ribosomal Protein L13 Participates in Innate Immune Response Induced by Foot-and-Mouth Disease Virus. Frontiers in Immunology, 12, 616402.

+ Open protocol

+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!