Monoclonal anti polyhistidine peroxidase antibody

Manufactured by Merck Group

Sourced in United States

Monoclonal anti-polyHistidine-Peroxidase antibody is a laboratory reagent used for the detection and quantification of proteins containing polyhistidine tags. It is a monoclonal antibody conjugated with the horseradish peroxidase enzyme, which can be used in various immunoassay techniques, such as Western blotting and ELISA, to enable colorimetric or chemiluminescent detection of target proteins.

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Lab products found in correlation

Amberlite xad4, by Merck Group (2 mentions) T4 dna ligase, by Thermo Fisher Scientific (2 mentions) Anti strep tag 2 antibody, by Abcam (1 mentions) 3 3 diaminobenzidine (dab), by Merck Group (1 mentions) Trans blot system, by Bio-Rad (1 mentions) Penta his antibody, by Qiagen (1 mentions) Monoclonal anti phosphotyrosine antibody, by Merck Group (1 mentions) Sephadex g 75, by Merck Group (1 mentions) Pcold 1 plasmid, by Takara Bio (1 mentions) L carnitine, by Merck Group (1 mentions) Protease inhibitor cocktail, by Merck Group (1 mentions) Phusion high fidelity dna polymerase, by Thermo Fisher Scientific (1 mentions) His select nickel affinity gel, by Merck Group (1 mentions) Lemo21 de3 strain, by New England Biolabs (1 mentions) N dodecyl β d maltoside, by Merck Group (1 mentions)

7 protocols using monoclonal anti polyhistidine peroxidase antibody

Protein Pulldown and Mass Spectrometry

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For pulldown experiments, 10 µM of either strep-tagged SbtB or His₆-tagged TrxA protein was incubated on either magnetic MagStrep “type3” XT beads (IBA GmbH, Göttingen) or Ni²⁺-NTA magnetic Beads (MagBeads; Genaxxon), respectively. For the detection of the protein, which coeluted with the immobilized protein, we used western blotting analysis using anti-Strep-tag II antibody (abcam) for detection of SbtB or monoclonal anti-polyHistidine-Peroxidase antibody (Sigma-Aldrich) for detection of TrxA.
To identify whether SbtB contains a protein contamination that possesses ATPase activity, recombinant purified SbtB protein from E. coli cells was-in gel digested with trypsin, then LC-MS/MS analysis was performed on a Proxeon Easy-nLC coupled to QExactive HF using 60 min gradient.

Selim K.A., Haffner M., Mantovani O., Albrecht R., Zhu H., Hagemann M., Forchhammer K, & Hartmann M.D. (2023). Carbon signaling protein SbtB possesses atypical redox-regulated apyrase activity to facilitate regulation of bicarbonate transporter SbtA. Proceedings of the National Academy of Sciences of the United States of America, 120(8), e2205882120.

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Immunohistochemical Evaluation of Anti-HER2 scFv

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The biological activity of anti-HER2 scFv refolded with optimum buffer composition, its ability to bind to HER2, was evaluated by immunohistochemistry staining. Breast cancer specimens, paraffin embedded sections, were incubated with refolded anti-HER2 scFv as the primary antibody. Monoclonal anti-polyhistidine-peroxidase antibody (Sigma, USA) and 3,3´diaminobenzidine (Sigma, USA) were used as the secondary antibody and the chromogen substrate, respectively. The sections were counterstained with hematoxilin.

Salehinia J., Sadeghi H.M., Abedi D, & Akbari V. (2018). Improvement of solubility and refolding of an anti-human epidermal growth factor receptor 2 single-chain antibody fragment inclusion bodies. Research in Pharmaceutical Sciences, 13(6), 566-574.

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Validation of Recombinant Protein Expression

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The presence and size of recombinant protein were checked on 12% SDS-PAGE and Western-Blot to validate fully expressed protein. The protein samples were loaded with Laemmli 4X (60 mM Tris–HCl pH 6.8, 10% glycerol, 2% SDS, 5% bromophenol Blue, 5% β- mercaptoethanol). A preliminary step of heating at 100 °C during 10 min was done before electrophoresis. The proteins were visualized by staining the gel with 0.2% Coomassie blue solution for 30 min and then destaining in a 20% ethanol and 10% acetic acid solution overnight, or transferred to Bio-Rad nitrocellulose membrane using a Trans-blot system (Bio-Rad) for Western blotting.
For Western-Blot, Nitrocellulose membranes were immersed in Tris-Buffer-Saline pH 7.6 0.1% Tween containing 5% milk for 30 min., then immersed in TBS-Tween-5% milk containing anti-his tag antibody (Monoclonal Anti-polyhistidine-Peroxidase antibody produced in mouse A7058, Sigma-Aldrich, Saint-Louis, MO, USA) at 1/5000 dilution for 45 min. Membrane was washed three times for 5 min in TBS-Tween. Detection was carried out using ECL™ Prime Western Blotting System (Cityva life sciences, Marlborough, USA). The protein marker used was Precision Plus Protein™ Prestained Standards all blue (BioRad).

Cochereau B., Le Strat Y., Ji Q., Pawtowski A., Delage L., Weill A., Mazéas L., Hervé C., Burgaud G., Gunde-Cimerman N., Pouchus Y.F., Demont-Caulet N., Roullier C, & Meslet-Cladiere L. (2023). Heterologous Expression and Biochemical Characterization of a New Chloroperoxidase Isolated from the Deep-Sea Hydrothermal Vent Black Yeast Hortaea werneckii UBOCC-A-208029. Marine Biotechnology (New York, N.y.), 25(4), 519-536.

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Antibody Detection and Characterization

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Monoclonal anti-polyHistidine-peroxidase antibody produced in mouse was purchased from Sigma (Cat. No. A7058). Monoclonal anti-phosphotyrosine antibody produced in mouse was purchased from Sigma (Cat. No. P4110). HRP conjugated anti-mouse IgG (H + L) antibody was purchased from Promega and used as the secondary antibody for detecting phosphotyrosine (Cat. No.W402B). AP-conjugated goat anti-rabbit IgG was purchased from Cedarlane (Cat. No. CLCC43008). Peroxidase-conjugated goat anti-mouse IgG was purchased from Cedarlane (Cat. No. 115-036-003). Penta-his antibody was purchased from Qiagen (Cat. No. 34660)

Yang Y., Liu J., Clarke B.R., Seidel L., Bolla J.R., Ward P.N., Zhang P., Robinson C.V., Whitfield C, & Naismith J.H. (2021). The molecular basis of regulation of bacterial capsule assembly by Wzc. Nature Communications, 12, 4349.

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Purification and Characterization of hCT2 Transporter

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Chemicals used for experiments, protease inhibitor cocktail (P8849), Nickel A nity Gel (HIS-Select® -P6611) and the Monoclonal Anti-polyHistidine-Peroxidase antibody (A7058) were from Sigma-Aldrich; pCOLD I plasmid from Takara; restriction endonucleases and speci c reagents for cloning from Thermo scienti c; E. coli RosettaGami2 strain from Novagen; BL21 codon plus RIL and Lemo21 from Agilent technologies; codon optimized hCT2 sequence was from Genscript. L-[ 3 H]-Carnitine from ARC (America Radiolabeled Chemicals), C 12 E 8 from TCI Europe, Amberlite XAD-4, egg yolk phospholipids, Sephadex G-75, L-carnitine from Sigma-Aldrich.

Galluccio M., Mazza T., Scalise M., Sarubbi M.C, & Indiveri C. (2022). Bacterial over-expression of functionally active human CT2 (SLC22A16) carnitine transporter. Molecular biology reports, 49(8).

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Quantitative Gene Expression and Protein Analysis

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Gene expression analysis was done by real-time PCR as follows. Total RNA was isolated from each sample and the expression levels of transcripts of all florescent genes in different strains were determined using primers listed in Table 1. rrsA (16S ribosomal RNA) gene was used for normalizing RNA expression.
Recombinant protein production was analyzed using Western blot. Total protein was isolated from each sample and separated using SDS-PAGE. Proteins were then transferred to nitrocellullose membrane, followed by immunodetection of proteins using monoclonal anti-poly histidine−peroxidase antibody from Merck.

Abdelaal A.S, & Yazdani S.S. (2021). A genetic toolkit for co-expression of multiple proteins of diverse physiological implication. Biotechnology Reports, 32, e00692.

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Heterologous Protein Expression and Purification

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His Select Nickel affinity gel (P6611), Monoclonal Anti-polyHistidine-Peroxidase antibody (A7058), Amberlite XAD-4 (06444), egg yolk phospholipids, cholesterol (C3045), Thrombin (605,157), n-Dodecyl β-D-maltoside (D4641), Sephadex G-75 (G75120), L-glutamine (G1626) from Merck Life Science; Isopropyl β-D-1-tiogalattopiranoside (IPTG, A4773) from AppliChem; restriction endonucleases, T4 DNA ligase (EL0014), and Phusion™ High-Fidelity DNA Polymerase (F530S) from ThermoFisher scientific; MegaMan Human Transcriptome Library, BL21 codon plus RIL strain from Agilent technologies; Lemo21(DE3) strain from New England Biolabs; C41(DE3) from Lucigen. Pico-Fluor Plus and L-[³H]-glutamine from Perkin Elmer; pET-28a-Mistic (#85,999) from Addgene.

Galluccio M., Tripicchio M., Console L, & Indiveri C. (2024). Bacterial over-production of the functionally active human SLC38A2 (SNAT2) exploiting the mistic tag: a cheap and fast tool for testing ligands. Molecular Biology Reports, 51(1), 336.

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