Horseradish peroxidase conjugated anti flag antibody

Manufactured by Merck Group

Sourced in United States

Horseradish peroxidase-conjugated anti-Flag antibody is a laboratory reagent used for the detection and identification of proteins or peptides that have been engineered to contain a specific amino acid sequence known as the Flag tag. The antibody is conjugated with the enzyme horseradish peroxidase, which can catalyze a color-producing reaction when exposed to a suitable substrate, allowing for the visualization and quantification of the tagged proteins.

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Lab products found in correlation

Horseradish peroxidase conjugated anti ha antibody, by Roche (3 mentions) Horseradish peroxidase conjugated goat anti mouse secondary antibody, by Bio-Rad (3 mentions) Horseradish peroxidase conjugated goat anti rabbit secondary antibody, by Bio-Rad (3 mentions) Horseradish peroxidase conjugated anti his6 antibody, by Merck Group (2 mentions) Anti cbp antibody, by Merck Group (2 mentions) Horseradish peroxidase conjugated anti rabbit secondary antibody, by Merck Group (2 mentions) Protease inhibitor cocktail, by Merck Group (2 mentions) Supersignal enzyme linked immunosorbent assay pico substrate, by Merck Group (2 mentions) Super signal enzyme linked immunosorbent assay pico substrate, by Thermo Fisher Scientific (2 mentions) Streptavidin hrp, by Thermo Fisher Scientific (1 mentions) Phos tag, by Fujifilm (1 mentions) Pflag cmv 4 expression vector, by Merck Group (1 mentions) Protein g sepharose bead, by GE Healthcare (1 mentions) Rabbit anti erk2, by Santa Cruz Biotechnology (1 mentions) Rabbit anti tubulin, by Cell Signaling Technology (1 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions) Cycloheximide (chx), by Merck Group (1 mentions) Mouse monoclonal anti β actin, by Merck Group (1 mentions) Mouse monoclonal anti flag antibody, by Merck Group (1 mentions) Mms 101r, by Merck Group (1 mentions)

Spelling variants of this product

Anti-FLAG (2121 citations) Anti-FLAG antibody (1016 citations) Horseradish peroxidase (696 citations) Flag antibody (199 citations) Peroxidase-conjugated anti-FLAG antibody (6 citations) Anti-Flag–horseradish peroxidase antibody (3 citations) Horseradish-conjugated anti-Flag antibody (2 citations) Horseradish Peroxidase conjugated anti-Flag (M2) antibody (2 citations) Horseradish peroxidase (HRP)-conjugated anti-FLAG antibody (2 citations)

15 protocols using horseradish peroxidase conjugated anti flag antibody

Western Blot Analysis of Ribosomal Proteins

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Western blot analysis was performed using the following antibodies: anti-Yar1 antibody (1:5,000; ref. 2222 (link)), anti-Rps3 antibody (1:30,000, provided by Matthias Seedorf), anti-Rps8 antibody (1:5,000, provided by Giorgio Dieci), anti-Rps2/Rpl30 antibody (1:2,000, provided by Jonathan Warner), anti-hRps5 antibody (1:500, Sigma-Aldrich, cat. no. HPA055878), anti-Rpl35 antibody (1:35,000, provided by Matthias Seedorf), anti-Rps26/Tsr2 antibody (1:2,000, provided by Vikram Panse), anti-Ltv1 antibody (1:8,000, provided by Katrin Karbstein), anti-Enp1 antibody (1:4,000, provided by Katrin Karbstein), anti-Tsr1 antibody (1:4,000, provided by Katrin Karbstein), anti-Dim1 antibody (1:4,000, provided by Katrin Karbstein), anti-Rio2-antibody (1:500, Santa Cruz Biotechnology, cat. no. sc-98828), anti-CBP antibody (1:4,000, Merck-Millipore, cat. no. 07-482), secondary anti-rabbit horseradish peroxidase-conjugated antibody (1:15,000, Sigma-Aldrich, cat. no. A0545), horseradish peroxidase-conjugated anti-His6 antibody (1:15,000, Sigma-Aldrich, cat. no. A7058), horseradish peroxidase-conjugated anti-Flag antibody (1:15,000, Sigma-Aldrich, cat. no. A8592).
The biotinylated phosphate-binding reagent Phos-Tag (Wako Pure Chemical Industries) was prepared according to manufacturer's instructions and used to detect protein phosphorylation in combination with streptavidin-HRP (Invitrogen).

Mitterer V., Murat G., Réty S., Blaud M., Delbos L., Stanborough T., Bergler H., Leulliot N., Kressler D, & Pertschy B. (2016). Sequential domain assembly of ribosomal protein S3 drives 40S subunit maturation. Nature Communications, 7, 10336.

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Comprehensive Western Blotting Protocol

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Western blot analysis was performed using the following antibodies: anti-Nog1 antibody (1:5000), anti-Nog2 antibody (1:20,000), anti-Arx1 antibody (1:2,000), anti-Nsa2 antibody (1:10,000), anti-Rlp24 antibody (1:2,000), provided by Micheline Fromont-Racine, anti-Nug1 antibody (1:10,000), anti-Bud20 antibody (1:5000), provided by Vikram Panse, anti-Ytm1 antibody (1:100), provided by John Woolford, anti-Has1 antibody (1:10,000) provided by Patrick Linder, anti-Ebp2 antibody (1:10,000), provided by Keiko Mizuta, anti-Noc3 antibody (1:1000), provided by Herbert Tschochner, anti-Rpl3 antibody (1:5,000), provided by Jonathan Warner, anti-Rsa4 antibody (1:10,000), provided by Miguel Remacha, anti-Arc1 antibody (1:5000, raised in the Hurt lab), horseradish-peroxidase-conjugated anti-Flag antibody (1:10,000; Sigma-Aldrich, Cat# A8592, RRID:AB_439702), horseradish-peroxidase-conjugated anti-HA antibody (1:5000; Roche, Cat# 12013819001; RRID:AB_390917), secondary horseradish-peroxidase-conjugated goat anti-rabbit antibody (1:2,000; Bio-Rad, Cat# 166-2408EDU, RRID:AB_11125345), and secondary horseradish-peroxidase-conjugated goat anti-mouse antibody (1:2,000; Bio-Rad, Cat# STAR105P, RRID:AB_323002).

Mitterer V., Thoms M., Buschauer R., Berninghausen O., Hurt E, & Beckmann R. (2023). Concurrent remodelling of nucleolar 60S subunit precursors by the Rea1 ATPase and Spb4 RNA helicase. eLife, 12, e84877.

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Detecting Human MX1 Protein

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Cell lysates from HEK293 cells transfected with pFLAG-CMV-4 expression vector (Sigma-Aldrich, St. Louis, MO, USA) encoding the full-length cDNA for human MX1 were immunoprecipitated using sera from representative patients and protein G–Sepharose beads (GE Healthcare, Buckinghamshire, UK). The precipitates were electrophoresed and detected with a horseradish peroxidase–conjugated anti-FLAG antibody (Sigma-Aldrich).

Hamano Y., Kida H., Ihara S., Murakami A., Yanagawa M., Ueda K., Honda O., Tripathi L.P., Arai T., Hirose M., Hamasaki T., Yano Y., Kimura T., Kato Y., Takamatsu H., Otsuka T., Minami T., Hirata H., Inoue K., Nagatomo I., Takeda Y., Mori M., Nishikawa H., Mizuguchi K., Kijima T., Kitaichi M., Tomiyama N., Inoue Y, & Kumanogoh A. (2017). Classification of idiopathic interstitial pneumonias using anti–myxovirus resistance-protein 1 autoantibody. Scientific Reports, 7, 43201.

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Characterization of FLT3 Receptor Mutants

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Human recombinant FLT3 ligand was from ORF genetics (Kópavogur, Iceland). The transfection reagent Lipofectamine 2000 was from Thermo Scientific and cycloheximide was from Sigma-Aldrich. pcDNA3-FLT3-WT, pMSCVpuro-FLT3-WT and pMSCVpuro-FLT3-ITD were described previously [18 (link)]. pMSCVpuro-FLT3-WT/Y842F and pMSCVpuro-FLT3-ITD/Y842F plasmids were generated by site-directed mutagenesis using QuikChange mutagenesis XL kit (Agilent Technologies). The anti-FLT3 antibody was a rabbit polyclonal antibody produced in-house. Mouse monoclonal anti-beta-actin, horseradish peroxidase-conjugated anti-FLAG antibody and mouse monoclonal anti-FLAG antibodies were from Sigma-Aldrich. Mouse anti-phosphotyrosine (4G10) antibody and mouse mono-ubiquitin antibody were from Millipore and Covance Research Products, respectively. Rabbit anti-ERK2, rabbit anti-phospho ERK1/2 (pThr202/pThr204), goat anti-AKT antibodies were from Santa Cruz Biotechnology. Rabbit anti-tubulin, rabbit anti-phospho-AKT (pSer473) rabbit anti-phospho GAB2 and rabbit anti-phospho-SHP2 were from Cell Signaling Technology.

Kazi J.U., Chougule R.A., Li T., Su X., Moharram S.A., Rupar K., Marhäll A., Gazi M., Sun J., Zhao H, & Rönnstrand L. (2017). Tyrosine 842 in the activation loop is required for full transformation by the oncogenic mutant FLT3-ITD. Cellular and Molecular Life Sciences, 74(14), 2679-2688.

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Antibody Panel for Western Blot Analysis

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Western blot analysis was performed using the following antibodies: anti-Nog1 antibody (1:5000), anti-Nog2 antibody (1:20,000), anti-Arx1 antibody (1:2000), anti-Rei1 antibody (1:10,000), anti-Nsa2 antibody (1:10,000), anti-Rlp24 antibody (1:2000), provided by Micheline Fromont-Racine, anti-Nug1 antibody (1:10,000), anti-Yvh1 antibody (1:4000), provided by Vikram Panse, anti-Nmd3 antibody (1:10,000), anti-Rpl10 antibody (1:10,000), provided by Arlen Johnson, anti-Rpl3 antibody (1:5000), provided by Jonathan Warner, anti-Rpl5 antibody (1:10.000), provided by John Woolford, anti-Nop7 antibody (1:50,000), provided by Bruce Stillman, anti-Rsa4 antibody (1:10,000), provided by Miguel Remacha, anti-Mrt4 antibody (1:1000), provided by Juan Pedro Ballesta, anti-Arc1 antibody (1:5000), raised in our lab, anti-HA antibody (1:10,000, Covance Research Products, MMS-101R), horseradish-peroxidase-conjugated anti-Flag antibody (1:15,000, Sigma–Aldrich, A8592), secondary horseradish-peroxidase-conjugated goat anti-rabbit antibody (1:2000, Bio-Rad-170-6515), secondary horseradish-peroxidase-conjugated goat anti-mouse antibody (1:2000, Bio-Rad-170-6516).

Thoms M., Mitterer V., Kater L., Falquet L., Beckmann R., Kressler D, & Hurt E. (2018). Suppressor mutations in Rpf2–Rrs1 or Rpl5 bypass the Cgr1 function for pre-ribosomal 5S RNP-rotation. Nature Communications, 9, 4094.

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Protein-Protein Interaction Analysis

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T o a n a l y z e p r o t e i n -p r o t e i n i n t e r a c t i o n s , immunoprecipitation-western blot analysis was performed as previously described (18) . Briefly, after transient co-expression of HA-tagged and Flag-tagged proteins, cells were lysed in lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% Nonidet P40, 0.25% sodium deoxycholate, 1 mM EDTA) containing Protease Inhibitor Cocktail (Sigma-Aldrich, St. Louis, MO, USA), immunoprecipitated with anti-HA antibody beads (Wako), and analyzed by western blotting using a horseradish peroxidase-conjugated anti-Flag antibody (Sigma-Aldrich). Total cell lysates were prepared and analyzed by western blotting as described previously ( 16), using anti-actin (1:2,000; A6050; Sigma-Aldrich), anti-p150 Glued (1:1,000; 610474; BD Biosciences, San Jose, CA, USA), anti-LC3 (1:2000; MBL, Nagoya, Japan), and anti-JLP (0.25 μg/mL (13)) primary antibodies.

Suzuki R., Gunarta I.K., Boldbaatar J., Erdenebaatar P., Odongoo R, & Yoshioka K. (2020). Functional role of c-Jun NH₂-terminal kinase-associated leucine zipper protein (JLP) in lysosome localization and autophagy. Drug discoveries & therapeutics, 14(1).

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Antibody-Based Protein Detection in Yeast

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Western blot analysis was performed using the following antibodies: anti-Nog1 antibody (1:5000), anti-Nog2 antibody (1:5000), anti-Nsa2 antibody (1:5000), anti-Rlp24 antibody (1:2000), provided by Micheline Fromont-Racine, anti-Nug1 antibody (1:10 000), anti-Bud20 antibody (1:5000), provided by Vikram Panse, anti-Has1 antibody (1:10 000) provided by Patrick Linder, anti-Ebp2 antibody (1:10 000), provided by Keiko Mizuta, anti-Rpl3 antibody (1:5000), provided by Jonathan Warner, anti-Rsa4 antibody (1:10 000), provided by Miguel Remacha, anti-Mrt4 antibody (1:1000) provided by Juan Pedro Ballesta, horseradish-peroxidase-conjugated anti-Flag antibody (1:10 000; Sigma-Aldrich, Cat# A8592, RRID:AB_439702), horseradish-peroxidase-conjugated anti-HA antibody (1:5000; Roche, Cat# 12013819001; RRID:AB_390917), secondary horseradish-peroxidase-conjugated goat anti-rabbit antibody (1:2000; Bio-Rad, Cat# 166-2408EDU, RRID:AB_11125345), secondary horseradish-peroxidase-conjugated goat anti-mouse antibody (1:2000; Bio-Rad, Cat# STAR105P, RRID:AB_323002).

Mitterer V., Hamze H., Kunowska N., Stelzl U., Henras A.K, & Hurt E. (2023). The RNA helicase Dbp10 coordinates assembly factor association with PTC maturation during ribosome biogenesis. Nucleic Acids Research, 52(4), 1975-1987.

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Protein Extraction and Western Blot Analysis

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Cells were inoculated in the desired medium with a starting OD₆₀₀ of 0.05. After 18 to 24 hours, the cells reached an OD₆₀₀ of 0.4 to 0.6/ml, and 10 OD₆₀₀ unit cells were collected and washed with sterile deionized water. The cell pellets were suspended in 250 μl of buffer A [2 M NaOH, 8% (v/v) β-mercaptoethanol] and incubated on ice for 5 min. The pellets were centrifuged at 6000g for 2 min at 4°C. Extracts were washed once with 500 μl of wash buffer [10 mM Hepes-KOH (pH 7.8), 300 mM NaCl, and 0.1% NP-40]. The cell extracts were resuspended and boiled with 200 μl of 1× sample buffer and boiled for 5 min. The samples were loaded onto a 4 to 20% gradient SDS gel (Invitrogen). The gel was electro-transferred to Immobilon-FL membranes (Millipore). The protein levels of Rad52 truncates were examined by probing with horseradish peroxidase–conjugated anti-Flag antibody (1:2000, Sigma-Aldrich).

Li F., Lee M., Esnault C., Wendover K., Guo Y., Atkins P., Zaratiegui M, & Levin H.L. (2022). Identification of an integrase-independent pathway of retrotransposition. Science Advances, 8(26), eabm9390.

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Western Blot Analysis of Ribosomal Proteins

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Western blot analysis was performed using the following antibodies: anti-Yar1 antibody (1:5,000)22 (link), anti-Rps3 antibody (1:30,000, provided by Matthias Seedorf), anti-Rps8 antibody (1:5,000, provided by Giorgio Dieci), anti-CBP antibody (1:4,000, Millipore), secondary anti-rabbit horseradish peroxidase-conjugated antibody (1:15,000, Sigma), anti-importin α antibody (1:200, Santa Cruz Biotechnology, sc-32681), anti-Kap95 antibody (1:200, Santa Cruz Biotechnology, sc-27053), secondary anti-goat horseradish peroxidase-conjugated antibody (1:12,000, Sigma), horseradish peroxidase-conjugated anti-His6 antibody (1:10,000, Sigma), horseradish peroxidase-conjugated anti-Flag antibody (1:10,000, Sigma), horseradish peroxidase-conjugated anti-HA antibody (1:5,000, Roche).

Mitterer V., Gantenbein N., Birner-Gruenberger R., Murat G., Bergler H., Kressler D, & Pertschy B. (2016). Nuclear import of dimerized ribosomal protein Rps3 in complex with its chaperone Yar1. Scientific Reports, 6, 36714.

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Protein Extraction and Western Blot Analysis

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Leaves were ground to a fine powder in liquid nitrogen and thawed in extraction buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10% [v/v] glycerol, 10 mM DTT, 10 mM EDTA, 1 mM NaF, 1 mM Na₂MoO₄·2H₂O, 1% [v/v] IGEPAL CA-630 from Sigma-Aldrich, and 1% [v/v] protease inhibitor cocktail from Sigma-Aldrich). Samples were cleared by centrifugation at 16,000 × g for 15 min at 4°C, and the supernatant was collected and subjected to SDS-PAGE. Proteins were then electroblotted onto a PVDF membrane using a semidry blotter (Trans-Blot Turbo Transfer System; Bio-Rad). Membranes were blocked overnight at 4°C in TBS-T (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.05% [v/v] Tween 20) with 5% (w/v) skim milk. Membranes were then incubated with horseradish peroxidase-conjugated anti-FLAG antibody (1:20,000; A8592; Sigma-Aldrich) diluted with TBS-T with 5% (w/v) skim milk at room temperature for 1 h. After washing with TBS-T, bound antibodies were visualized using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific). Bands were imaged using an image analyzer (ImageQuant LAS 4000 imager; GE Healthcare).

Asai S., Cevik V., Jones J.D, & Shirasu K. (2023). Cell-specific RNA profiling reveals host genes expressed in Arabidopsis cells haustoriated by downy mildew. Plant Physiology, 193(1), 259-270.

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