Peroxidase conjugated secondary antibody

Manufactured by Proteintech

Sourced in United States, China

Peroxidase-conjugated secondary antibody is a laboratory reagent used in various immunoassay techniques, such as Western blotting and enzyme-linked immunosorbent assay (ELISA). It is designed to detect and visualize primary antibodies that have been bound to their target antigens. The peroxidase enzyme attached to the secondary antibody catalyzes a colorimetric reaction, allowing for the detection and quantification of the target protein.

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Lab products found in correlation

Polyvinylidene difluoride membrane, by Bio-Rad (7 mentions) β actin, by Proteintech (6 mentions) Gapdh, by Proteintech (4 mentions) Protease inhibitor cocktail, by Thermo Fisher Scientific (3 mentions) E cadherin, by Cell Signaling Technology (2 mentions) Pvdf membrane, by Merck Group (2 mentions) Ripa lysis buffer, by Beyotime (2 mentions) Pvdf membrane, by Cytiva (2 mentions) Enhanced chemiluminescence kit, by GE Healthcare (2 mentions) Anti gapdh antibody, by Proteintech (2 mentions) Vimentin, by Santa Cruz Biotechnology (2 mentions) E cadherin, by Santa Cruz Biotechnology (2 mentions) Rabbit anti plasminogen antibody, by Proteintech (2 mentions) Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions) Ab32536, by Abcam (2 mentions) Amersham imager, by Cytiva (2 mentions) Cell lysis buffer, by Cell Signaling Technology (1 mentions) Enhanced chemiluminescence, by Merck Group (1 mentions) Cell counting kit 8 cck 8 kit, by Solarbio (1 mentions) Vascular endothelial growth factor (vegf), by Cell Signaling Technology (1 mentions)

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25 protocols using peroxidase conjugated secondary antibody

Immunoprecipitation and Immunoblotting Techniques

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Immunoprecipitation and immunoblotting were performed according to previously reported methods.⁵² (link)^,⁵⁶ (link) The cells were lysed with cell lysis buffer (Cell Signaling Technology), which was supplemented with a protease inhibitor cocktail (Calbiochem). The protein concentrations of the extracts were measured using a bicinchoninic acid assay (Pierce).
Immunoprecipitation was performed according to our previously published method.⁵⁹ (link) The gut epithelial cells were lysed in immunoprecipitation lysis buffer (Pierce, Rockford, IL) containing 10% phenylmethylsulfonyl fluoride. Protein A/G magnetic beads (Pierce) were first added into the cell lysates for preclearing. The supernatant was collected after centrifuging at 12,000 rpm and then immunoprecipitated overnight at 4°C with the antibodies or control antibodies. Protein A/G magnetic beads were added into the cell lysates and incubated for an additional 3 hours. After being washed 5 times, the lysates were denatured and resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis.
For immunoblotting, hybridizations with primary antibodies were conducted for 1 hour at room temperature in blocking buffer. The protein–antibody complexes were detected using peroxidase-conjugated secondary antibodies (Proteintech) and enhanced chemiluminescence (Millipore).

Zhang Q., Su X., Zhang C., Chen W., Wang Y., Yang X., Liu D., Zhang Y, & Yang R. (2022). Klebsiella pneumoniae Induces Inflammatory Bowel Disease Through Caspase-11–Mediated IL18 in the Gut Epithelial Cells. Cellular and Molecular Gastroenterology and Hepatology, 15(3), 613-632.

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Investigating NKP608 Anticancer Mechanisms

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NKP608 was purchased from Medchem Express (New Jersey, USA). RPMI-1640 was supplied from Hyclone Laboratories (Logan, UT, USA). Fetal bovine serum (FBS) was from Gibco Cell Culture (Carlsbad, CA, USA). Cell counting kit-8 (CCK8) kit was obtained from Beijing Solarbio Science and Technology (Beijing, China). Metrigel was supplied with BD Biosciences (MA, USA). Annexin V-fluorescein isothiocyanate/propidium iodide (Annexin V-FITC/PI) Apoptosis Detection kit was from Beijing 4A Biotech (Beijing, China). Radioimmunoprecipitation assay (RIPA) lysis buffer and bicinchoninic acid (BCA) assay was from cwbiotech (Beijing, China).
Antibodies to Active-Caspase-3, Wnt-3a, β-catenin, E-Cadherin, (vascular endothelial growth factor) VEGF were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). Other antibodies to Cyclin D1, Bcl-2, Bax, GAPDH and peroxidase conjugated secondary antibodies were provided from Proteintech Group, Inc. (Wuhan, China). The enhanced chemiluminescence (ECL) detection system was obtained from Proteintech Group, Inc. (Wuhan, China).

Niu X.L., Hou J.F, & Li J.X. (2018). The NK1 receptor antagonist NKP608 inhibits proliferation of human colorectal cancer cells via Wnt signaling pathway. Biological Research, 51, 14.

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Protein Transfer and Immunoblotting

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Trans‐Blot Transfer system (Bio‐Rad Laboratories) was used to transfer proteins to PVDF membranes. The PVDF membranes were then blocked with soluble 5% dry milk in 1x TBST (Tris‐buffered saline with 0.1% Tween‐20) for 1–2 h at room temperature and then incubated with primary antibody at 4°C overnight. The antibodies included TNFa, MMP‐3, MCP‐1(1:1000 dilution, Cell signalling technology), a‐tubulin (1:1000 dilution, Proteintech), and peroxidase‐conjugated secondary antibodies (1:1000 dilution, Proteintech). The PVDF membranes were then incubated with secondary antibodies. Images were obtained with image lab software using ChemiDoc charge‐coupled device (CCD) camera (Bio‐Rad Laboratories).

Wang Z., Chai C., Wang R., Feng Y., Huang L., Zhang Y., Xiao X., Yang S., Zhang Y, & Zhang X. (2021). Single‐cell transcriptome atlas of human mesenchymal stem cells exploring cellular heterogeneity. Clinical and Translational Medicine, 11(12), e650.

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Western Blot Analysis of Apoptosis Markers

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The proteins from SKOV-3 and T47D cells or tumor tissues were extracted by ice-cold RIPA lysis buffer (Solarbio, China) added with protease inhibitor cocktail (Thermo), and separated by 8–10% SDS-PAGE. The separated proteins were transferred to NC membranes. The blots were blocked and incubated with specific primary antibodies against GPX4 (1:1000, Proteintech, China), SLC7A11 (1:1000, Proteintech), Bcl-2 (1:1000, abcam, United States), Bax (1:1000, abcam, United States), caspase3 (1:1000, abcam, United States), cleaved-caspase3 (1:1000, abcam, United States), RIPK3 (1:1000, abcam, United States), and β-actin (1:1000, Proteintech) overnight at 4°C. Following washing with PBST, the blots were incubated at room temperature by peroxidase-conjugated secondary antibodies (1:1000, Proteintech). Proteins were visualized by using an ECL reagent (Millipore, United States) in an Image Lab Software (BIO RAD, United States).

Sun D., Li Y.C, & Zhang X.Y. (2021). Lidocaine Promoted Ferroptosis by Targeting miR-382-5p /SLC7A11 Axis in Ovarian and Breast Cancer. Frontiers in Pharmacology, 12, 681223.

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Quantitative Analysis of Protein Expression

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Total protein was extracted using a Total Protein Extraction Kit (Nanjing KeyGen Biotech Co., Ltd.), and the protein concentration was quantified by using a BCA Protein Assay Kit (Nanjing KeyGen Biotech Co., Ltd.), according to the manufacturer's instructions. Subsequently, 30 µg of protein was separated using 10% SDS-PAGE and then electrophoretically transferred to PVDF membranes (MilliporeSigma). The membranes were incubated at room temperature for 2 h with 5% non-fat dry milk in Tris-buffered saline-0.5% Tween-20 prior to immunoblotting with primary antibodies against MEF2C (1:1,000; cat. no. YT2702, ImmunoWay Biotechnology Company) and TCF4 (1:1,000; cat. no. YT4580; ImmunoWay Biotechnology Company) at 4°C overnight. Following incubation with 1:10,000 donkey anti-mouse (cat. no. sa00001-1) or donkey anti-rabbit (cat. no. sa00001-9) peroxidase-conjugated secondary antibodies (Wuhan Sanying Biotechnology; ProteinTech Group, Inc.) for 2 h at room temperature, the antigen-antibody complexes were visualized using the FluorChem 2.01 System (Alpha Innotech Corporation; ProteinSimple) and an Enhanced Chemiluminescence Kit (Pierce; Thermo Fisher Scientific, Inc.). Protein levels in miR-190b mimic-transfected AsPC-1 cells are presented as fold-change values relative to the levels in NC-transfected AsPC-1 cells after normalization to GAPDH as an endogenous reference.

Li Y., Wang Z., Zhao F., Zeng J, & Yang X. (2021). MicroRNA-190b expression predicts a good prognosis and attenuates the malignant progression of pancreatic cancer by targeting MEF2C and TCF4. Oncology Reports, 47(1), 12.

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Apoptosis Signaling Pathway Antibodies

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All primary antibodies utilized in this study included Bax (1:1000), Bcl2 (1:1000), Bad (1:1000), Caspase-3 (1:1000), Cyt C (1:1500), Cleaved-Caspase9 (1:1500), Cleaved-Caspase-3 (1:1000, all from Cell Signaling Technology, USA), p-p53 (1:1000), p53 (1:1000, Abcam, UK), PEDV N (1:1000), GAPDH (1:5000), β-actin (1:5000), and VDAC-1 (1:3000, ProteinTech Group, Wuhan, China). Secondary antibodies were peroxidase-conjugated secondary antibodies (1:1500, ProteinTech Group, Wuhan, China).

Wang X., Liu Y., Li K., Yang M., Wang Q, & Hao Z. (2022). Triacetyl Resveratrol Inhibits PEDV by Inducing the Early Apoptosis In Vitro. International Journal of Molecular Sciences, 23(23), 14499.

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Immunoblotting Analysis of Bacterial Toxin

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Bacterial culture was performed as described in the hemolysis assay. After the A600nm reached 2.1, equal volumes of supernatants of different samples were harvested by centrifugation (2 400 g, 5 minutes). Next, 5×SDS‐PAGE loading buffer containing β‐mercaptoethanol（β‐Me）was added, and the mixture was boiled for 10 minutes (100°C). Samples was separated by 12% SDS‐PAGE, and the proteins were transferred onto polyvinylidene fluoride（PVDF）membranes. After blocking the membranes in a blocking buffer (5% BSA) for 2 h at room temperature, the membranes were first incubated with primary rabbit antibody against LLO (Abcam, diluted with blocking buffer at 1:1000) and then a peroxidase‐conjugated secondary antibody (Proteintech, diluted with blocking buffer at 1:5000). The protein band signals were visualized with a Tanon‐4200 imager, using ECL reagent (Pierce™ ECL Western Blotting Substrate).

Lu G., Xu L., Zhang T., Deng X, & Wang J. (2018). A potential bio‐control agent from baical skullcap root against listeriosis via the inhibition of sortase A and listeriolysin O. Journal of Cellular and Molecular Medicine, 23(3), 2042-2051.

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Western Blot Analysis of Bladder Cancer Cells

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Bladder cancer cells were washed with ice‐cold phosphate buffered saline (PBS), lysed in RIPA lysis buffer supplemented with protease and phosphatase inhibitors, and the concentration was measured by BCA Protein Assay Kit (Invitrogen).
¹⁸ (link),
¹⁹ (link) For western blot analysis, 30 μg of proteins were loaded onto 10% SDS‐PAGE and transferred to PVDF membranes. Next, the membranes were blocked with 5% milk for 1 h at room temperature, and incubated with anti‐FLRT2 (#ab154023, Abcam), anti‐ACSL4 (#ab155282, Abcam) or anti‐β‐actin (#ab8226, Abcam) overnight at 4°C. On the next day, the sample was then washed thrice with TBST for 10 min each time. The membrane was removed and incubated with the peroxidaseconjugated secondary antibody (ProteinTech Group) at 37°C for 2 h. Finally, ECL (Biosharp Life Sciences) colour was developed with β‐actin as an internal reference to analyse the protein expression level on the membrane and visualized with the by the chemiluminescence system (ChemiDocTM Touch; Bio‐Rad).

Jiang P., Ning J., Yu W., Rao T., Ruan Y, & Cheng F. (2023). FLRT2 suppresses bladder cancer progression through inducing ferroptosis. Journal of Cellular and Molecular Medicine, 28(5), e17855.

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Western Blot Protein Detection

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Protein extracts were resolved via 8% SDS-PAGE, transferred onto polyvinylidene difluoride membranes (BioRad, Berkeley, CA), probed with antibodies against human integrin β3 (Abcam, Cambridge, UK), p-FAK (Y397), FAK, p-Src (Y416), Src (Cell signaling, Boston, MA) or β-actin (Proteintech, Chicago, IL) followed by a peroxidase-conjugated secondary antibody (Proteintech), and then visualized by chemiluminescence (ImageQuant RT ECL, GE, Fairfield, CT).

Sun L., Liu B., Lin Z., Yao Y., Chen Y., Li Y., Chen J., Yu D., Tang Z., Wang B., Zeng S., Fan S., Wang Y., Li Y., Song E, & Li J. (2015). MiR-320a acts as a prognostic factor and Inhibits metastasis of salivary adenoid cystic carcinoma by targeting ITGB3. Molecular Cancer, 14, 96.

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Western Blot Analysis of EMT Markers

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Total cell lysates were obtained using RIPA Lysis Buffer (Beyotime, Shanghai, China) supplemented with 1:25 Halt Phosphatase Inhibitor Cocktail (Roche, Shanghai, China), and protein concentration was determined using BCA Protein Assay Kit (Pierce, Grand Island, NY, USA). Protein samples were prepared to 20 μg per well, denatured in 100 °C for 10 min, separated by 10% SDS/PAGE, and then transferred to a PVDF membrane (Millipore, Shanghai, China). Membranes were immunoblotted with primary antibodies, anti‐PNN (Proteintech, Wuhan, China), anti‐E‐cadherin, anti‐N‐cadherin, anti‐ZEB1, anti‐ZEB2, anti‐SNAIL, or anti‐SLUG (CST, Danvers, MA, USA) antibodies. After washing, the blots were incubated with respective peroxidase‐conjugated secondary antibody (Proteintech, Wuhan, China). The blot was exposed to X‐ray film and developed using an enhanced chemiluminescence detection system (EMD Millipore, Burlington, MA, USA). GAPDH (CST, Danvers, MA, USA) served as the loading control.

Tang T., Yang L., Cao Y., Wang M., Zhang S., Gong Z., Xiong F., He Y., Zhou Y., Liao Q., Xiang B., Zhou M., Guo C., Li X., Li Y., Xiong W., Li G, & Zeng Z. (2020). LncRNA AATBC regulates Pinin to promote metastasis in nasopharyngeal carcinoma. Molecular Oncology, 14(9), 2251-2270.

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