Anti flag hrp antibody

Manufactured by Merck Group

Sourced in Canada

The Anti-FLAG-HRP antibody is a detection reagent used in biochemical and molecular biology applications. It is conjugated to horseradish peroxidase (HRP), which allows for sensitive and specific detection of target proteins carrying a FLAG tag. The antibody recognizes the FLAG epitope, a short peptide sequence commonly used as a protein tag for affinity purification and immunodetection.

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Lab products found in correlation

Anti rabbit igg hrp, by Thermo Fisher Scientific (2 mentions) Anti cmyc hrp antibody, by Merck Group (2 mentions) Pvdf membrane, by Merck Group (2 mentions) Rabbit anti actin antibody, by Santa Cruz Biotechnology (2 mentions) Anti rabbit rgs1 antibody, by Thermo Fisher Scientific (2 mentions) Rabbit anti cox 4, by Cell Signaling Technology (1 mentions) Anti rabbit igg hrp linked antibody, by Cell Signaling Technology (1 mentions) Normal rabbit igg, by Cell Signaling Technology (1 mentions) Mouse anti β actin a5316, by Merck Group (1 mentions) N ethylmaleimide, by Merck Group (1 mentions) Protease inhibitor cocktail, by Merck Group (1 mentions) Anti streptavidin hrp, by Cell Signaling Technology (1 mentions) Protein a g coupled agarose beads, by Thermo Fisher Scientific (1 mentions) Pip strip, by Echelon Biosciences (1 mentions) Donkey anti igg rabbit hrp polyclonal antibodies, by BioLegend (1 mentions) Anti gfp hrp antibodies, by Santa Cruz Biotechnology (1 mentions) Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (1 mentions) Ab167453, by Abcam (1 mentions) Anti gfp hrp antibody, by Miltenyi Biotec (1 mentions) N myc tagged pmx puro retroviral vector, by Cell Biolabs (1 mentions)

Spelling variants of this product

Anti-FLAG (2121 citations) Anti-FLAG antibody (1016 citations) Flag antibody (199 citations) Anti-FLAG-HRP (103 citations) Flag-HRP (41 citations) ANTI-FLAG® M2-Peroxidase (HRP) antibody (29 citations) Anti-FLAG M2 HRP-conjugated monoclonal antibody (3 citations) Mouse monoclonal anti‐FLAG M2‐HRP antibody (2 citations) Monoclonal anti-FLAG antibody conjugated to HRP (2 citations) Mouse-anti-Flag-HRP antibody (2 citations)

18 protocols using anti flag hrp antibody

Antibody Validation for Immunoblotting

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Mouse anti-β-actin (A5316) and anti-Flag-HRP antibodies (A8592) were purchased from Sigma-Aldrich. Anti-Myc-horseradish peroxidase (HRP) antibody (#2040), anti-HA-HRP antibody (#2999), rabbit anti-IRF-3 (#11904), rabbit anti-pIRF-3 (#4947), rabbit anti-Flag (#14793), rabbit anti-LDHA (#3582), rabbit anti-COX IV (#4844), anti-rabbit IgG-HRP-linked antibody (#7074), anti-mouse IgG-HRP-linked antibody (#7076), normal rabbit IgG (#2729) were bought from Cell-Signaling Technologies. Mouse anti-Myc antibody (sc-40), mouse anti-HA antibody (sc-7392), mouse anti-RIG-I (sc-376845), mouse anti-14-3-3ε-horseradish peroxidase (HRP) antibody (sc-23957 HRP), mouse anti-V5 antibody (sc-81594), mouse anti-MAVS (sc-365334), normal mouse IgG (sc-2025) were purchased from Santa Cruz Biotechnology. Mouse anti-HSV ICP27 antibody (P1113) was purchased from Virusys. Anti-γ₁34.5 and NS1 antibodies were described previously [79 (link),80 (link)].

Liu X., Ma Y., Voss K., van Gent M., Chan Y.K., Gack M.U., Gale M J.r, & He B. (2021). The herpesvirus accessory protein γ134.5 facilitates viral replication by disabling mitochondrial translocation of RIG-I. PLoS Pathogens, 17(3), e1009446.

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Detecting PPARγ SUMOylation in Cells

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HEK293 cells transfected with BLRP-tagged PPARγWT or PPARγSUMO (K107, 395R), biotin-protein ligase (BirA) and Flag-tagged SUMO1, or HBEC cells infected with BirA adenovirus were lysed in immunoprecipitation (IP) lysis buffer containing 10 mM tris-HCl pH 7.5, 150 mM NaCl, 10 mM phosphate buffer, 1% triton X-100, 20 mM N-ethylmaleimide (Sigma) and protease inhibitor cocktail (Roche). Cell lysates were immunoprecipitated overnight at 4°C with streptavidin resin beads (Thermo) and washed 5 times in IP washing buffer (phosphate-buffered saline with 0.5% NP40). Immunoprecipitates were resolved by SDS-PAGE and immunoblotted using anti-Streptavidin-HRP (Cell Signaling Technology, #3999) and anti-Flag-HRP antibodies (Sigma, #A8592).
For endogenous detection of sumoylated PPARγ, Calu6 cells treated with pioglitazone 30μM for 24 hours were harvested in IP lysis buffer, followed by incubation overnight at 4°C with SUMO1 antibody (#4940), IP with protein A/G-coupled agarose beads (Thermo) and 5 washes with IP washing buffer. Immunoprecipitates were then resolved by SDS-PAGE and immunoblotted using indicated antibodies.

Phan A.N., Vo V.T., Hua T.N., Kim M.K., Jo S.Y., Choi J.W., Kim H.W., Son J., Suh Y.A, & Jeong Y. (2017). PPARγ sumoylation-mediated lipid accumulation in lung cancer. Oncotarget, 8(47), 82491-82505.

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NLRP3 Lipid Binding Assay using PIP Strips

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Lipid binding assay with purified NLRP3 variants was performed using PIP strip (Echelon Biosciences, Cat. no: P-6001) according to manufacturer instructions. In short, the PIP strip membranes were blocked using 3% bovine serum albumin (BSA) in phosphate-buffered saline with 0.1% Tween 20 (PBS-T) for 1 h following by incubation with 30 μl NLRP3 FLAG-tagged proteins (0.5 μM) diluted in 3% BSA in PBS-T for 1 h. Next, the membranes were washed in PBS-T for 40 min and the bound proteins were visualized with anti-FLAG-HRP antibodies (1:10000 in PBS-T for 1h, Sigma-Aldrich, Cat. no: A8592). All steps were performed at room temperature. All samples were analysed at the same time under the same conditions.

Andreeva L., David L., Rawson S., Shen C., Pasricha T., Pelegrin P, & Wu H. (2021). NLRP3 Cages Revealed by Full-length Mouse NLRP3 Structure Control Pathway Activation. Cell, 184(26), 6299-6312.e22.

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Protein Extraction and Immunoblotting Analysis

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Protein extraction and analysis was carried out as previously described 15 . Proteins were detected by immune blotting using anti-HA-horseradish peroxidase conjugated (HRP) antibodies (Sigma, 1:3,000 dilution), anti-FLAG-HRP antibodies (Sigma, 1:5,000 dilution), anti-GFP-HRP antibodies (Santa Cruz, 1:3,000 dilution), anti-PVX-CP rabbit polyclonal antibodies (Agdia, 1:3,000 dilution) and anti-AGO2 antibody (Agrisera, 1:5,000 dilution or Abiocode 1:7,500). Detection of the latter three primary antibodies was performed using donkey anti-IgG rabbit-HRP polyclonal antibodies (BioLegend, 1:10,000 dilution).

Brosseau C., Bolaji A., Roussin-Léveillée C., Zhao Z., Biga S, & Moffett P. (2020). Natural variation in the Arabidopsis AGO2 gene is associated with susceptibility to potato virus X. The New phytologist, 226(3).

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Protein Quantification in Plant Tissues

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To determine FLAG-RGA, Myc-SPY and Myc-SEC protein levels in Arabidopsis or tobacco tissues, total proteins were extracted and analyzed by SDS-PAGE and immunoblotting as described^{48 (link)}. An anti-cMyc-HRP antibody (Sigma-Aldrich, Cat. A5598; 8,000× dilution) was used to detect Myc-SPY. A monoclonal anti-FLAG-HRP antibody (Sigma-Aldrich, Cat. A8592; 10,000× dilution) was used to detect FLAG-RGA. An anti-SEC antisera^{20 (link)} (2,000× dilution) and anti-rabbit IgG-HRP (Thermo-Fisher, Cat. 31462; 20,000× dilution) were used to detect Myc-SEC. Affinity-purified polyclonal anti-SPY antibodies (anti-LQKE^{49 (link)}; 6,000× dilution), and anti-rabbit IgG-HRP (same as above) were used to detect recombinant WT and mutant SPY proteins for the in vitro enzyme assays, and for Myc-SPY in the tobacco samples.

Zentella R., Sui N., Barnhill B., Hsieh W.P., Hu J., Shabanowitz J., Boyce M., Olszewski N.E., Zhou P., Hunt D.F, & Sun T.P. (2017). The Arabidopsis O-fucosyltransferase SPINDLY activates nuclear growth repressor DELLA. Nature chemical biology, 13(5), 479-485.

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Protein Quantification in Plant Tissues

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Western Blot Analysis of Protein Expression

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Proteins were transferred to nitrocellulose membrane (GE Healthcare, Amersham^TM Highbond^TM-ECL) via wet western blotting at 4 °C and 30 V overnight. Membranes were blocked in TBS-Tween (0.1%)–Milk (5%) for 1 h at room temperature. For mCherry detection, membranes were incubated overnight with an anti-mCherry antibody (ab167453, dilution 1 : 2000; Abcam) followed by 1 h incubation with an anti-rabbit-horseradish peroxidase (HRP) antibody (dilution 1 : 10,000, Calbiochem). For GFP detection, membranes were incubated overnight with an anti-GFP-HRP antibody (130-091-833, dilution 1 : 1000, Miltenyi Biotec). For FLAG detection, membranes were incubated overnight with an anti-FLAG-HRP antibody (A8692, dilution 1 : 1000, Sigma). For SPX1 detection, membranes were incubated overnight with an anti-SPX1 antibody (dilution 1 : 1000, kind gift of Professor Mingguang Lei) followed by one hour incubation with an anti-rabbit-HRP antibody (dilution 1 : 10,000, Calbiochem). Antibodies were diluted in TBS-Tween (0.05%)–Milk (2.5%). Membranes were detected with SuperSignal™ West Femto Maximum Sensitivity Substrate (34095, Thermo Scientific™).

Ried M.K., Wild R., Zhu J., Pipercevic J., Sturm K., Broger L., Harmel R.K., Abriata L.A., Hothorn L.A., Fiedler D., Hiller S, & Hothorn M. (2021). Inositol pyrophosphates promote the interaction of SPX domains with the coiled-coil motif of PHR transcription factors to regulate plant phosphate homeostasis. Nature Communications, 12, 384.

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Engineered NOTCH1-ROS1 Fusion Constructs

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NOTCH1–ROS1 and Trunc. NOTCH1–ROS1 constructs were synthesized as gateway compatible entry clones by GenScript. Full-length NOTCH1 and full-length ROS1 were purchased as gateway entry clones from GenScript and PlasmID/Dana-Farber/Harvard Cancer Center DNA Resource Core, respectively. Subcloning was conducted to integrate NOTCH1–ROS1, Trunc. NOTCH1–ROS1, NOTCH1, and ROS1 into Gateway-compatible N-MYC-tagged pMX-Puro Retroviral Vector (Cell Biolabs). NIH3T3 cells were transduced using a previously described infection protocol (Jain et al. 2017 (link)). Ba/F3 cells were transduced with virus generated from the Plat-E retroviral packaging cell line (Cell Biolabs). Prior to infection, cells were treated with 10 µg/mL polybrene. Virus was harvested at 48 h and filtered with a 0.45 µm syringe filter before addition. Upon viral infection and addition of 10 mM HEPES, cells were inoculated for 90 min and then incubated for 72 h. An amount of 2 µg/mL puromycin was then utilized to select stably transduced cells. All constructs were subcloned into gateway destination vectors with amino-terminal Myc and Flag tags Invitrogen). Expression of tagged proteins was detected using anti-Myc HRP Antibody (Invitrogen R951-25; 1:5000) and anti-Flag HRP antibody (Sigma-Aldrich A8592; 1:5000).

Jain P., Iyer S., Straka J., Surrey L.F., Pogoriler J., Han H., Smith T., Busch C., Fox E., Li M., Waanders A.J., Resnick A, & Davare M.A. (2022). Discovery and functional characterization of the oncogenicity and targetability of a novel NOTCH1–ROS1 gene fusion in pediatric angiosarcoma. Cold Spring Harbor Molecular Case Studies, 8(6), a006222.

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Quantification of UbV-E3 Ligase Binding

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Proteins in study were immobilized on 384-well MaxiSorp plates (Thermo Scientific, Mississauga, ON, Canada 12665347) by adding 30 µL of 1 µM proteins for overnight incubation at 4 °C. Phage and protein ELISA against immobilized proteins was performed as described [16 (link)]. Binding of phages was detected using anti-M13-HRP antibody (GE Healthcare, Mississauga, ON, Canada 27942101) and binding of FLAG-tagged UbVs was detected using anti-FLAG-HRP antibody (Sigma-Aldrich, Oakville, Canada A8592). To measure the half maximal binding concentration (EC₅₀) of UbVs binding to E3 ligases, the concentration of UbVs or wild type Ub was varied from 0 to 4 µM (24 points, 1:2 dilution), while the concentration of target proteins immobilized on the plate remained at 1 µM. EC₅₀ values were calculated using the GraphPad Prism software with the built-in equation formula (non-linear regression curve).

Aminu B., Fux J., Mallette E., Petersen N, & Zhang W. (2022). Targeted Degradation of 53BP1 Using Ubiquitin Variant Induced Proximity. Biomolecules, 12(4), 479.

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Quantification of FLAG-tagged NOCT Protein

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Protein concentrations for HEK293A cells overexpressing FLAG-tagged NOCT were determined using the BCA assay (ThermoFisher Scientific #23225), then the lysates were diluted to 5mg/mL with RIPA buffer and adjusted a final concentration of 1mg/mL with 5X Laemmli sample buffer. Each well on a 4–20% Trisglycine (Bio-Rad #4568096) gel was loaded with 10μg of total protein and SDS-PAGE was performed at 80V for 2 hours. Proteins were transferred to a PVDF membrane at 100V for 1 hour using wet transfer. Blocking was done in 5% milk/TBST for 1 hour at room temperature. Membranes were incubated in anti-FLAG-HRP antibody (Sigma #A8592) at 1:10,000 dilution in 5% milk/TBST for 1 hour at room temperature. For secondary antibody incubation, membranes were incubated with anti-rabbit HRP (Cell Signaling #7074S) at 1:2,000 dilution in 5% milk/TBST for 1 hour at room temperature. Membranes were washed with TBST multiple times. ECL reaction was performed using Bio-Rad’s Clarity Max (Bio-Rad #1705062) for 5 minutes, and the chemiluminescence signal was measured on the ChemiDoc MP imager.

Wickramaratne A.C., Li L., Hopkins J.B., Joachimiak L.A, & Green C.B. (2022). The disordered amino terminus of the circadian enzyme Nocturnin modulates its NADP(H) phosphatase activity by changing protein dynamics. Biochemistry.

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