α flag m2 hrp

Manufactured by Merck Group

The α-FLAG-M2-HRP is a laboratory reagent that binds to the FLAG peptide tag. It is conjugated with horseradish peroxidase (HRP) enzyme, which can be used for detection in various immunoassay and Western blotting applications.

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Lab products found in correlation

Amersham imager 600, by GE Healthcare (1 mentions) Amersham ecl prime western blotting detection reagent, by GE Healthcare (1 mentions) α tubulin hrp, by Santa Cruz Biotechnology (1 mentions) Hrp coupled α mouse, by GE Healthcare (1 mentions) α gfp, by Roche (1 mentions) α myc, by Fortrea (1 mentions) Amersham imager, by Cytiva (1 mentions) Amersham ecl prime, by Cytiva (1 mentions) Polyvinylidene difluoride membrane, by Merck Group (1 mentions) Enhanced chemiluminescence detection, by Merck Group (1 mentions) Gapdh 6c5, by Thermo Fisher Scientific (1 mentions) Chemidoc digital imaging system, by Bio-Rad (1 mentions) Image lab package, by Bio-Rad (1 mentions) Any kd mini protean tgx precast gel, by Bio-Rad (1 mentions) Pvdf membrane, by Bio-Rad (1 mentions) Immobilon forte western hrp substrate, by Merck Group (1 mentions) Expressplus page 4 20 gels, by GenScript (1 mentions) Mouse α β actin, by Merck Group (1 mentions) Trans blot turbo transfer system, by Bio-Rad (1 mentions) Bioruptor, by Diagenode (1 mentions)

Close spelling variants of this product

FLAG M2 (383 citations) α-FLAG (120 citations) Flag-HRP (41 citations) α-FLAG M2 (40 citations) α-FLAG-HRP (9 citations)

6 protocols using α flag m2 hrp

Comprehensive Antibody Detection Protocol

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Antibodies used were α-GFP (Roche; from mouse, catalog no. 11814460001) 1:1,000; α-myc (Covance; 9E10 from mouse, catalog no. MMS-150R) 1:1,000; α-PGK 1:10,000 (Invitrogen; from mouse, catalog no. 459250); α-Flag M2-HRP (Sigma; from mouse, catalog no. A 8592) 1:10,000; α-tubulin-HRP 1:1,000 (Santa Cruz; from rat, catalog no. sc-53030); and HRP-coupled α-mouse (GE Healthcare; catalog no. NA931) 1:10,000. All antibodies were diluted in 5% (wt/vol) milk in Tris-buffered saline containing 0.01% (vol/vol) Tween-20.
For visualization Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare) was used. Images were acquired by Amersham Imager 600 (GE Healthcare).

Kornakov N., Möllers B, & Westermann S. (2020). The EB1–Kinesin-14 complex is required for efficient metaphase spindle assembly and kinetochore bi-orientation. The Journal of Cell Biology, 219(12), e202003072.

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Quantitative Analysis of MeV Proteins

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BSR-T7/5 cells were transfected in a 12-well plate format (4 × 10⁵ per well) with 2 μg of N-encoding plasmid DNA, and 40 hours after transfection, cells were washed once with phosphate-buffered saline (PBS) and lysed in radioimmunoprecipitation assay buffer [1% sodium deoxycholate, 1% NP-40, 150 mM NaCl, 50 mM tris-Cl (pH 7.2), 10 mM EDTA, 50 mM NaF, 0.05% SDS, protease inhibitors (Roche), 1 mM phenylmethylsulfonyl fluoride]. Cleared lysates (20,000g, 10 min, 4°C) were mixed with 5× urea buffer [200 mM tris (pH 6.8), 8 M urea, 5% SDS, 0.1 mM EDTA, 0.03% bromphenol blue, 1.5% dithiothreitol]. Samples were denatured for 30 min at 50°C, fractionated on 8% SDS–polyacrylamide gel electrophoresis (PAGE) gels, blotted on polyvinylidene difluoride membranes (Millipore), and subjected to enhanced chemiluminescence detection (Pierce) using specific antibodies directed against MeV-N (83KKKII, Millipore), MeV-P (9H4, Abcam), GAPDH (6C5, Ambion), α-FLAG-M2-HRP (Sigma-Aldrich), or HA (16B12, Sigma-Aldrich), as specified. Immunoblots were developed using a ChemiDoc digital imaging system (Bio-Rad). Only nonsaturated images were used for densitometry and carried out using the Image Lab package (Bio-Rad) and global background correction.

Cox R.M., Krumm S.A., Thakkar V.D., Sohn M, & Plemper R.K. (2017). The structurally disordered paramyxovirus nucleocapsid protein tail domain is a regulator of the mRNA transcription gradient. Science Advances, 3(2), e1602350.

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Identification of ORMM1 Interacting Proteins

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1% of the IP samples were loaded onto an Any kD MINI-PROTEAN TGX precast gel (Bio-Rad, Hercules, CA) followed by standard procedures of immunoblotting. α-FLAG-M2-HRP (Sigma-Aldrich, St. Louis, MO) was used to detect FLAG-tagged proteins. 10% of the IP samples were separated on a 10% polyacrylamide gel before being subjected to silver staining compatible to mass spectrometry. Co-purifying proteins with FLAG-tagged ORMM1 were identified by tandem mass spectrometry using a nanoLC-LTQ-Orbitrap instrument, followed by database searching with MASCOT against TAIR10 [36 (link)].

Sun T., Shi X., Friso G., Van Wijk K., Bentolila S, & Hanson M.R. (2015). A Zinc Finger Motif-Containing Protein Is Essential for Chloroplast RNA Editing. PLoS Genetics, 11(3), e1005028.

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Western Blot Protein Analysis

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15 to 30 µg of total protein samples were resolved on ExpressPlus™ PAGE 4-20% gels (GenScript), in MOPS running buffer, or 8% Tris-glycine polyacrylamide gels in SDS/Tris-glycine running buffer, according to the size-resolution required. The resolved proteins were transferred to polyvinylidenedifluoride (PVDF) membranes (Bio-Rad), through a Trans-Blot® Turbo™ Transfer System (Bio-Rad). PVDF membranes were subsequently blocked in Tris-buffered saline plus 0.1% Tween (TBS-T) and 5% non-fat milk for 1 hour at 4°C and then incubated over-night at 4°C with primary antibodies in TBS-T plus 5% non-fat milk. Membranes were then washed in TBS-T (3x10 minutes) at room temperature (RT) and subsequently incubated for 1 hour at RT with horseradish peroxidase (HRP)-conjugated α-mouse or α-rabbit IgG. Blots were then washed in TBS-T (4x10 min) at RT and rinsed in TBS-T, and immunoreactive proteins were visualised using Immobilon® Forte Western HRP Substrate (Merck Millipore). Densitometric analysis was carried out using the ImageJ software. The antibodies used for Western Blot are as follows: mouse α-β-actin (A1978, Sigma-Aldrich, 1:20000), mouse α-pan 14-3-3 (sc-1657, Santa Cruz Biotechnology, 1:1000 to 1:10000), α-Flag® M2-HRP (A8592, Sigma-Aldrich, 1:20000), mouse α-His-HRP (A7058, Sigma-Aldrich, 1:10000).

Cogo S., Tomkins J.E., Vavouraki N., Giusti V., Forcellato F., Franchin C., Tessari I., Arrigoni G., Cendron L., Manzoni C., Civiero L., Lewis P.A., & Greggio E. (2020). Cytosolic sequestration of spatacsin by Protein Kinase A and 14-3-3 proteins.

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Immunoprecipitation and Western Blot Protocol

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Cells were harvested and washed twice in PBS before lysing them in 150 mM KAc pH 7.4, 30 mM HEPES pH 7.4, 5 mM MgAc, 1 mM DTT, 15% glycerol, 1% tergitol and 1 tablet of protease inhibitor per 10 ml (complete mini EDTA-free, Roche). Lysis was further facilitated using a Bioruptor (Diagenode, 20 cyles: 20 s ON, 20 s OFF). The lysate was cleared by centrifugation before it was incubated with Protein G Dynabeads (Invitrogen) that had been coated with 1 μl of anti-FLAG M2 (Sigma) per 10 μl beads. The beads were washed twice with 150 mM KAc pH 7.4, 30 mmM HEPES pH 7.4, 5 mM MgAc, 0.1% tergitol and twice with 150 mM KAc pH 7.4, 30 mmM HEPES pH 7.4, 5 mM MgAc. The beads were boiled in SDS loading dye for analysis on a SDS-PAGE and subsequent Western Blot performed as previously described (36 (link)). Antibodies used for the Western Blot were α-FLAG-HRP M2 (Sigma), α-Strep-tag II-HRP (IBA) or anti-Dcr2 (a kind gift of M. Siomi).
Details on our immunoprecipitations for mass spectrometry are given in the supplementary information.

Nitschko V., Kunzelmann S., Fröhlich T., Arnold G.J, & Förstemann K. (2020). Trafficking of siRNA precursors by the dsRBD protein Blanks in Drosophila. Nucleic Acids Research, 48(7), 3906-3921.

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Western Blot Protein Detection

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Samples were resolved on 8% Tris/glycine polyacrylamide gels and transferred to a PDVF membrane (Millipore Immobilon, Merck, pore size 0.45 μm). The membranes were blocked by 5% non‐fat dry milk powder (NFDM) in TBS‐T (20 mM Tris–HCl pH 7.4, 150 mM NaCl and 0.1% (v/v) Tween‐20) for at least 1 h at RT. Primary antibodies were diluted in 5 % NFDM in TBS‐T as follows: α‐HA‐4D2 (Roche) 1:2,000; α‐PTH1R 4D2 (Thermo Fisher Scientific) 1:2,000; α‐FLAG‐HRP M2 (Sigma‐Aldrich) 1:5,000; α‐1D4‐HRP (Santa Cruz Biotechnology, sc‐57432) 1:2,000. Membranes were incubated for either 1 h at RT (α‐FLAG‐HRP) or overnight at 4°C with the primary antibody (α‐HA, α‐PTH1R and α‐1D4‐HRP), followed by 3 × 10 min washes in TBS‐T. Secondary antibodies, either α‐rat‐HRP (Cell Signaling) or α‐mouse‐HRP (Santa Cruz Biotechnology, sc‐516102), were used at 1:10,000 in 5% NFDM in TBS‐T for 1 h at RT followed by 3 × 10 min wash cycles in TBS‐T. Membranes were soaked in homemade ECL reagent (10 parts 0.1 M Tris–HCl pH 8.6 with 250 mg/l luminol, one part DMSO with 1,100 mg/l p‐hydroxycoumarin acid, and 0.003 parts 30% H₂O₂). After 1 min, signals were detected for 5 min in the dark (Gbox, Syngene).

Böttke T., Ernicke S., Serfling R., Ihling C., Burda E., Gurevich V.V., Sinz A, & Coin I. (2020). Exploring GPCR‐arrestin interfaces with genetically encoded crosslinkers. EMBO Reports, 21(11), e50437.

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