Horseradish peroxidase conjugated antibody

Manufactured by Jackson ImmunoResearch

Sourced in United States

Horseradish peroxidase-conjugated antibodies are laboratory reagents used in various immunoassay techniques. They consist of antibodies that have been chemically conjugated to the enzyme horseradish peroxidase. These conjugated antibodies can be used to detect and quantify target analytes in samples.

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Lab products found in correlation

Micro bca protein assay kit, by Thermo Fisher Scientific (2 mentions) Fitc phalloidin, by Merck Group (2 mentions) Anti gm130 antibody, by BD (2 mentions) Anti flag m2 and anti vinculin antibodies, by Merck Group (2 mentions) Phospho akt ser473 and phospho erk1 2 thr202 tyr204 antibodies, by Santa Cruz Biotechnology (2 mentions) Akt and erk1 2 antibodies, by Cell Signaling Technology (2 mentions) 2 conjugated anti goat igg h l and rhodaminered x conjugated anti mouse igg h l antibodies, by Jackson ImmunoResearch (2 mentions) Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (2 mentions) Guinea pig anti insulin, by Merck Group (2 mentions) Re blot plus strong solution, by Merck Group (2 mentions) Phosstop, by Merck Group (2 mentions) Complete protease inhibitor cocktail, by Merck Group (2 mentions) Nupage gel, by Thermo Fisher Scientific (2 mentions) Bovine serum albumin (bsa), by Merck Group (2 mentions) Fuji las3000, by Fujifilm (1 mentions) Phospho iκbα ser32 14d4, by Cell Signaling Technology (1 mentions) Mab374, by Merck Group (1 mentions) Cx40 a, by Alpha Diagnostic (1 mentions) Ubiquitin, by Santa Cruz Biotechnology (1 mentions) Cyp2e1, by Abcam (1 mentions)

23 protocols using horseradish peroxidase conjugated antibody

Western Blot Analysis of Signaling Proteins

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Cell cultures were rinsed with PBS, pH=7.4, and lysed in RIPA buffer as previously described [15 (link)]. After protein concentration quantification with a Micro BCA protein assay kit (Thermo Scientific), an equal quantity of protein was separated by SDS-PAGE and transferred to PVDF-membrane (Immobilon Millipore). After 2 hours blocking with 5% milk and 1% Tween in PBS, the membrane was exposed to primary antibodies (in blocking solution) detecting Cx40 (Cx40-A; Alpha-Diagnostics lot #175455A8.6; 1/500), NFκB (NFκB p65 (A) sc-109; Santa Cruz; 1/1000), P-NFκB (Phospho-NFκB (S536)(93H1); Cell Signaling; 1/1000), IκBα (IκBα L35A5; Cell Signaling; 1/1000), P-IκBα (Phospho-IκB-α (Ser32)(14D4); Cell Signaling; 1/1000) and GAPDH (Millipore MAB374; lot #2388833; 1/30000) as loading control. Revelation was performed by incubating the membrane for 1 hour at RT with secondary horseradish peroxidase-conjugated antibodies (Jackson Immunoresearch; 1/5000) and followed by ECL detection (Millipore) using the Fuji LAS3000 (Fujifilm) and ImageQuant LAS 4000 software. Band intensities were thereafter quantified using ImageJ software.

Denis J.F., Scheckenbach K.E., Pfenniger A., Meens M.J., Krams R., Miquerol L., Taffet S., Chanson M., Delmar M, & Kwak B.R. (2017). Connexin40 controls endothelial activation by dampening NFκB activation. Oncotarget, 8(31), 50972-50986.

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Monoclonal Antibodies for Actinin-4 and Actinin-1

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Mouse monoclonal antibodies recognizing both α-actinin-4 and -1 were prepared using a GST fusion protein containing α-actinin-4 residues 311–911 as an antigen based on our previously described method ^{26 (link), 27 (link)}. The following antibodies were purchased: anti-CLP36 antibody (Imegenex); antibody recognizing α-actinin-1 but not -4 (Abnova); anti-FLAG M2 and anti-vinculin antibodies (Sigma); Phospho-Akt (Ser473) and Phospho-Erk1/2 (Thr202/Tyr204) antibodies (Santa Cruz Biotech); Akt and Erk1/2 antibodies (Cell Signaling); anti-GM130 antibody (BD biosciences); ^™2-conjugated anti-Goat IgG (H+L) and RhodamineRed^™-X-conjugated anti-mouse IgG(H+L) antibodies and horseradish peroxidase-conjugated antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA). FITC-phalloidin was from Sigma. Chemiluminescent substrate was from Millipore (Catalog number WBKLS0500). In some Western blotting experiments, a SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific product number 34096) was used.

Liu Z., Zhan Y., Tu Y., Chen K., Liu Z, & Wu C. (2014). PDZ and LIM domain protein 1(PDLIM1)/CLP36 promotes breast cancer cell migration, invasion and metastasis through interaction with α-actinin. Oncogene, 34(10), 1300-1311.

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Monoclonal Antibodies for Actinin-4 and Actinin-1

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Western Blot Analysis of Oxidative Stress

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The following antibodies were used for western blot analysis: Parkin (Santa-Cruz, SC-32282), Ubiquitin (Santa Cruz, SC-8017), p62 (Abnova, H00008878-M01), β-Actin (Sigma, A5441), Cyp2e1 (Abcam, ab19140), phosphorylated JNK (4668S), JNK (BD, 554285), Oxphos rodent antibody cocktail (Abcam, ab110413), and voltage-dependent anion channel (VDAC) (Calbiochem, 529534). The APAP-adduct antibody was a gift from Dr. Lance Pohl (NIH) [22 (link)]. Horseradish peroxidase-conjugated antibodies were from Jackson ImmunoResearch Lab. Adenovirus (Ad) Cox8-GFP-mCherry was produced in collaboration with Vector Biolabs (Malvern, PA). In situ cell death detection kit (Cat# 11684809910) was purchased from Roche. The kit for alanine aminotransferase (ALT) assay was purchased from Pointe Scientific (A7526-450). APAP and other chemicals were either purchased from Sigma-Aldrich or Thermo Fisher Scientific.

Wang H., Ni H.M., Chao X., Ma X., Rodriguez Y.A., Chavan H., Wang S., Krishnamurthy P., Dobrowsky R., Xu D.X., Jaeschke H, & Ding W.X. (2019). Double deletion of PINK1 and Parkin impairs hepatic mitophagy and exacerbates acetaminophen-induced liver injury in mice. Redox Biology, 22, 101148.

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Western Blot Protein Analysis Protocol

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Protein extracts were prepared as for native gel electrophoresis. For Western blotting, proteins were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto a 0.45 mm PVDF membrane. Membranes were blocked overnight at 4 °C with 5% milk in phosphate-buffered saline (PBS) and 0.5% Tween-20 (PBST). Membranes were incubated for 60 min with primary antibodies (antibody details in SI Appendix) at room temperature. Primary antibodies were detected by secondary species-specific horseradish peroxidase-conjugated antibodies (1:5,000; Jackson ImmunoResearch). Detection was performed with Amersham ECL Western Blotting Detection Reagent.

Minis A., Rodriguez J.A., Levin A., Liu K., Govek E.E., Hatten M.E, & Steller H. (2019). The proteasome regulator PI31 is required for protein homeostasis, synapse maintenance, and neuronal survival in mice. Proceedings of the National Academy of Sciences of the United States of America, 116(49), 24639-24650.

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Cx40 Protein Expression Analysis

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Cell cultures were rinsed with PBS, pH = 7.4, and lysed in RIPA buffer as previously described (Pfenniger et al., 2012 (link)). After protein concentration quantification with a Micro BCA protein assay kit (Thermo Scientific), 5 μg (HUVECs) or 10 μg (bEnd.3) of protein was separated by SDS-PAGE and transferred to PVDF-membrane (Immobilon, Millipore). After 2 h blocking with 5% milk and 1% Tween in PBS, the membrane was exposed to anti-Cx40 (Alpha-Diagnostics, 1/500) or anti-β-actin (Sigma, 1/10000) primary antibodies in blocking solution. Revelation was performed by incubating the membrane for 1 h at RT with secondary horseradish peroxidase-conjugated antibodies (Jackson ImmunoResearch; 1/5000) and followed by ECL detection (Millipore) using ImageQuant LAS 4000 software. Band intensities were thereafter quantified using ImageJ software. Cx40 results were normalized to β-actin.

Denis J.F., Diagbouga M.R., Molica F., Hautefort A., Linnerz T., Watanabe M., Lemeille S., Bertrand J.Y, & Kwak B.R. (2019). KLF4-Induced Connexin40 Expression Contributes to Arterial Endothelial Quiescence. Frontiers in Physiology, 10, 80.

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Insulin Signaling Pathway Evaluation

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Lipofectamine 2000 for transfection, Lipofectamine RNAiMAX, 4–12% NuPage gels, LDS sample loading buffer, Met/Cys-deficient Dulbecco’s modified Eagle’s medium, and all other tissue culture reagents were from Invitrogen (Carlsbad, CA, USA). The siRNAs were from ThermoFisher (Waltham, MA, USA). The rodent (mouse/rat) insulin chemiluminescence ELISA kit was from ALPCO company (Salem, NH, USA). Protein A-Agarose was from Repligen (Waltham, MA, USA). ³⁵S-amino acid mixture was from PerkinElmer (Waltham, MA). Rabbit anti-SSR1 antibody and mouse anti-rat proinsulin antibody CCI-17 were from Novus Biologicals (Littleton, CO, USA). Guinea pig anti-insulin was from Merck-Millipore (Billerica, MA, USA). Rabbit anti-SSR2 was from Proteintech (Rosemont, IL, USA). Rabbit polyclonal anti-SSR3 and anti-SSR4 were from Abmart (Shanghai, China). Mouse monoclonal anti-tubulin was from Sigma (St. Louis, MO, USA). Horseradish peroxidase-conjugated antibodies were from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). Clarity Western ECL Substrate was from Bio-Rad (Hercules, CA, USA).

Huang Y., Xu X., Arvan P, & Liu M. (2021). Deficient endoplasmic reticulum translocon-associated protein complex limits the biosynthesis of proinsulin and insulin. FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 35(5), e21515.

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Lipofectamine 2000 Transfection Protocol

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Lipofectamine 2000 and 4–12% NuPage gel were purchased from Invitrogen (Carlsbad, CA, USA). Protein phosphatase inhibitor was purchased from Beyotime Biotechnology (Beijing, China). Protein A-Agarose was from Santa Cruz Biotechnology (Dallas, TX, USA). Guinea pig anti-insulin (dilution: 1:2000) was from Merck Millipore (Billerica, MA, USA) and mouse anti-proinsulin antibody was from Novus Biologicals (Littleton, CO, USA). Rabbit anti-Hsp90 (dilution: 1:2000) antibody was from Assay designs (Ann Arbor, MI, USA). Rabbit anti-cleaved caspase 3 antibody was from Cell Signalling Technology (Danvers, MA, USA). Annexin V with Alexa Fluor™ 555 conjugation, Goat anti-guinea pig IgG Alexa Fluor 555 and goat anti-rabbit IgG Alexa Fluor 435 was bought from Invitrogen (Carlsbad, CA, USA). Rabbit anti-Myc antibody was from Immunology Consultants Labs. Horseradish peroxidase–conjugated antibodies were from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). Enhanced chemiluminescence Western blotting substrate was from Millipore (Billerica, MA, USA). Trans³⁵S label and pure ³⁵S-methionine were from PerkinElmer (Waltham, MA, USA).

Wang H., Saint-Martin C., Xu J., Ding L., Wang R., Feng W., Liu M., Shu H., Fan Z., Haataja L., Arvan P., Bellanné-Chantelot C., Cui J, & Huang Y. (2020). Biological Behaviors of Mutant Proinsulin Contribute to the Phenotypic Spectrum of Diabetes Associated with Insulin Gene Mutations. Molecular and cellular endocrinology, 518, 111025.

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Western Blot Protein Detection

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Protein samples were obtained from cell lysates using lysis buffer (#9803; Cell Signaling) containing PhosSTOP (#04-906-845-001; Merck) and Complete Protease Inhibitor Cocktail (#11-697-498-001; Merck). Protein samples were reduced with β-mercaptoethanol or dithiothreitol and heated at 95°C with shaking. Samples were loaded on 10% SDS–Page and run at 120 V. Protein samples were transferred onto polyvinylidene fluoride (PVDF) membranes previously activated with methanol. After blocking for 1 h with 4% BSA (#A7906; Merck), the membrane was incubated overnight with primary antibodies diluted in 4% BSA. GAPDH was used for normalisation. Horseradish peroxidase-conjugated antibodies (Jackson Immuno Research) were used as secondary antibodies with incubation for 1 h. Western Lightning Plus-ECL Enhanced Chemiluminescence (NEL105001EA; Perkin Elmer) was used as substrate to develop the membrane in a colorimetric or fluorescence detection. Image SXM or Fusion FX7Edge software were used to quantify protein expression. For multiple stainings, the membranes were re-blotted after stripping with Re-Blot Plus Strong Solution (#2504; Merck).

Della Verde G., Mochizuki M., Lorenz V., Roux J., Xu L., Ramin-Wright L., Pfister O, & Kuster G.M. (2021). Fms-like tyrosine kinase 3 is a regulator of the cardiac side population in mice. Life Science Alliance, 5(3), e202101112.

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Immunohistochemical Analysis of β-Catenin

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For immunohistochemistry, sections were quenched with 3% H₂O₂/H₂O. Antigen retrieval was performed in a 10 mM sodium citrate buffer and sections were preincubated with donkey serum (10% in PBS). Sections were incubated overnight at 4°C with primary antibody against β-catenin (1:200 dilution; Santa Cruz). Negative control studies were performed with species-specific IgG (Jackson ImmunoResearch). Secondary antibodies were horseradish peroxidase-conjugated antibodies (1:200 dilution; Jackson ImmunoResearch).

De Langhe E., Aznar-Lopez C., De Vooght V., Vanoirbeek J.A., Luyten F.P, & Lories R.J. (2014). Secreted frizzled related proteins inhibit fibrosis in vitro but appear redundant in vivo. Fibrogenesis & Tissue Repair, 7, 14.

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