Anti flag m2 hrp antibody

Manufactured by Merck Group

Sourced in United States

The Anti-FLAG M2-HRP antibody is a conjugated antibody that binds to the FLAG epitope tag. It is used for the detection and purification of FLAG-tagged proteins in various applications.

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Lab products found in correlation

Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions) Ab22512, by Abcam (1 mentions) Ecl western blotting detection reagent, by GE Healthcare (1 mentions) Las 4000, by Fujifilm (1 mentions) Polyvinylidene difluoride membrane, by Bio-Rad (1 mentions) Anti α tubulin antibody, by Abcam (1 mentions) Rhodamine phalloidin, by Cytoskeleton (1 mentions) Anti flag antibody, by Cell Signaling Technology (1 mentions) Alexa fluor secondary antibody, by Thermo Fisher Scientific (1 mentions) Rabbit anti lc3 antibody, by Novus Biologicals (1 mentions) Mouse anti gapdh antibody, by Merck Group (1 mentions) Anti beclin 1 antibody, by Santa Cruz Biotechnology (1 mentions) Mouse anti p62 antibody, by Abnova (1 mentions) Rabbit anti tom20 antibody, by Santa Cruz Biotechnology (1 mentions) Phosphatase inhibitor, by Merck Group (1 mentions) Protease inhibitor cocktail, by Merck Group (1 mentions) Affinipure goat anti mouse igg h l, by Jackson ImmunoResearch (1 mentions) Precellys evolution homogenizer, by Bertin Technologies (1 mentions) Tet repressor, by Thermo Fisher Scientific (1 mentions)

9 protocols using anti flag m2 hrp antibody

Antibody Detection Protocol

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The antibodies used were anti-FLAG M2-HRP antibody (A8592; Sigma), anti-SGN5
(sc-13157; Santa Cruz, Paso Robles, CA), anti-tubulin (MS-581-P0; NeoMarkers, Fremont,
CA), anti-WD repeat-containing protein 5 (anti-WDR5) (ab22512, Abcam, Cambridge, England),
anti-WDR62 (A301–560A; Bethyl, Montgomery, TX), anti-LIS1 antibody (AB5413,
Millipore, Billerica, MA), anti-RBX1, and anti-DDB1 (both generated as described
previously [Ohta et al., 1999 (link); Hu et al., 2004 (link)]).

Vulto-van Silfhout A.T., Nakagawa T., Bahi-Buisson N., Haas S.A., Hu H., Bienek M., Vissers L.E., Gilissen C., Tzschach A., Busche A., Müsebeck J., Rump P., Mathijssen I.B., Avela K., Somer M., Doagu F., Philips A.K., Rauch A., Baumer A., Voesenek K., Poirier K., Vigneron J., Amram D., Odent S., Nawara M., Obersztyn E., Lenart J., Charzewska A., Lebrun N., Fischer U., Nillesen W.M., Yntema H.G., Järvelä I., Ropers H.H., de Vries B.B., Brunner H.G., van Bokhoven H., Raymond F.L., Willemsen M.A., Chelly J., Xiong Y., Barkovich A.J., Kalscheuer V.M., Kleefstra T, & de Brouwer A.P. (2015). Variants in CUL4B are Associated with Cerebral Malformations. Human mutation, 36(1), 106-117.

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Stable HEK293 Cell Line Generation

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For stable HEK293 cells, cells were cultivated as indicated above and transfected with the corresponding DNA construct using PEI reagent. After 48 h, the medium was exchanged by growing medium supplemented with G418 (Biochrom, 750 mg ml⁻¹). The cells were cultivated for ∼3 weeks, until the transiently transfected cells died. The medium was exchanged regularly to ensure normal growth. Afterwards, the cells were split in a ratio of 1:100 and cultivated until single colonies were observed. Colonies were transferred to six-well plates and cultivated to confluency. For evaluation, a part of the cells was lysed and applied to western blot analysis, using an anti-FLAG-M2-HRP antibody (Sigma-Aldrich) for detection of the expressed SF-TAP-fusion protein.

Boldt K., van Reeuwijk J., Lu Q., Koutroumpas K., Nguyen T.M., Texier Y., van Beersum S.E., Horn N., Willer J.R., Mans D.A., Dougherty G., Lamers I.J., Coene K.L., Arts H.H., Betts M.J., Beyer T., Bolat E., Gloeckner C.J., Haidari K., Hetterschijt L., Iaconis D., Jenkins D., Klose F., Knapp B., Latour B., Letteboer S.J., Marcelis C.L., Mitic D., Morleo M., Oud M.M., Riemersma M., Rix S., Terhal P.A., Toedt G., van Dam T.J., de Vrieze E., Wissinger Y., Wu K.M., Apic G., Beales P.L., Blacque O.E., Gibson T.J., Huynen M.A., Katsanis N., Kremer H., Omran H., van Wijk E., Wolfrum U., Kepes F., Davis E.E., Franco B., Giles R.H., Ueffing M., Russell R.B, & Roepman R. (2016). An organelle-specific protein landscape identifies novel diseases and molecular mechanisms. Nature Communications, 7, 11491.

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Western Blot Protein Detection Protocol

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The stacking gel was composed of 12% (wt/vol) acrylamide, 0.1% (wt/vol) sodium dodecyl sulfate (SDS), 125 mM Tris–HCl (pH 6.8), 0.1% (wt/vol) ammonium persulfate, and 0.5% (vol/vol) N,N,N′,N′‐tetramethylethylenediamine. The resolving gel was composed of 12% (wt/vol) acrylamide, 0.1% (wt/vol) SDS, 375 mM Tris–HCl (pH 8.8), 0.05% (wt/vol) ammonium persulfate, and 0.25% (wt/vol) N,N,N′,N′‐tetramethylethylenediamine. Electrophoresis was performed in 0.1% (wt/vol) SDS, 25 mM tris, and 192 mM glycine at 180 V per gel for 50 min. Proteins were transferred to poly(vinylidene difluoride) membranes (0.2 μm; Bio‐Rad) at 15 V for 30 min in transfer buffer. Membranes were incubated with 3% (wt/vol) non‐fat dry milk in Tris‐buffered saline containing Tween [TTBS; 50 mM Tris–HCl (pH 7.8), 150 mM NaCl, and 0.1% (vol/vol) Tween 20] for 1–2 h and then in anti‐FLAG M2‐HRP antibody (1:4,000; Sigma‐Aldrich, A8592) in TTBS for 2 h at room temperature. After washing with TTBS (three times for 5 min each), membranes were incubated with the ECL Western Blotting Detection Reagent (GE Healthcare). The emitted light was detected and quantified with a chemiluminescence imaging analyzer (LAS4000, Fujifilm), and images were analysed with Adobe Photoshop Elements 13.

Hodatsu A., Fujino N., Uyama Y., Tsukamoto O., Imai‐Okazaki A., Yamazaki S., Seguchi O., Konno T., Hayashi K., Kawashiri M., Asano Y., Kitakaze M., Takashima S, & Yamagishi M. (2019). Impact of cardiac myosin light chain kinase gene mutation on development of dilated cardiomyopathy. ESC Heart Failure, 6(2), 406-415.

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Immunostaining and Immunoblotting Protocol

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Antibodies used were anti-FLAG antibodies (1:400 dilution for immunostaining, no. 14793; Cell Signaling Technology, Danvers, MA, USA); Alexa Fluor secondary antibodies (1:500 dilution for immunostaining, Thermo Fisher Scientific, Waltham, MA, USA); rhodamine phalloidin (3:500 dilution for immunostaining, PHDR1; Cytoskeleton, Inc., Denver, CO, USA); anti-FLAG^® M2-HRP antibody (1:4,000 dilution for immunoblotting, A8592; Sigma–Aldrich, St. Louis, MO, USA); anti-GFP HRP-DirecT antibody (1:2000 for immunoblotting, no. 598-7; MBL, Nagoya, Japan); anti-α-tubulin antibody (1:1000 for immunoblotting, no. B-5-1-2; Abcam, Cambridge, UK); HRP-coupled goat anti-mouse antibody (1:8,000, no. 55550; MP biomedicals, Santa Ana, CA, USA); and anti-skeletal RLC known as MYLPF (1:4000, dilution for immunoblotting, 16052-1-AP; Proteintech, Rosemont, IL, USA).

Oya R., Tsukamoto O., Hitsumoto T., Nakahara N., Okamoto C., Matsuoka K., Kato H., Inohara H, & Takashima S. (2022). Gene Transfer of Skeletal Muscle-Type Myosin Light Chain Kinase via Adeno-Associated Virus 6 Improves Muscle Functions in an Amyotrophic Lateral Sclerosis Mouse Model. International Journal of Molecular Sciences, 23(3), 1747.

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Western Blot Analysis of Autophagy

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Flag was detected using an anti-Flag M2 HRP antibody (1:2000) (Sigma-Aldrich, St. Louis, MO). The phosphospecific Beclin 1 S90 antibody was produced by PhosphoSolutions (Aurora, CO). Briefly, synthetic peptides corresponding to phosphorylated and dephosphorylated S90 of Beclin 1 were injected to two rabbits and sera were purified using a phosphopeptide affinity column followed by a dephosphopeptide affinity column. The phosphospecific Beclin 1 S90 antibody was used at a concentration of 1:500. Beclin 1, HSP27, p-HSP27, Actin, MK2, p62, LC3, TOM20, PDI, and GAPDH were detected using a rabbit or goat anti-Beclin 1 antibody (Santa Cruz Biotechnology, Dallas, TX, 1:1000 dilution), a rabbit anti-HSP27 antibody (Santa Cruz Biotechnology, 1:200 dilution), a rabbit anti-p-HSP27 antibody (Santa Cruz Biotechnology, 1:200 dilution), an anti-β-Actin HRP antibody (Santa Cruz Biotechnology, 1:2000 dilution), a rabbit anti-MK2 antibody (Cell Signaling Technology, Beverly, MA; 1:1000 dilution), a mouse anti-p62 antibody (Abnova, Walnut, CA; 1:2000 dilution) a rabbit anti-LC3 antibody (Novus Biologicals, Littleton, CO; 1:1000 dilution), a rabbit anti-TOM20 antibody (Santa Cruz Biotechnology; 1:1000 dilution), a rabbit anti-PDI antibody (Cell Signaling Techology; 1:1000 dilution), and a mouse anti-GAPDH antibody (Chemicon International, Temecula, CA; 1:000 dilution).

Wei Y., An Z., Zou Z., Sumpter R J.r., Su M., Zang X., Sinha S., Gaestel M, & Levine B. (2015). The stress-responsive kinases MAPKAPK2/MAPKAPK3 activate starvation-induced autophagy through Beclin 1 phosphorylation. eLife, 4, e05289.

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CD44-MSN Interaction Assay

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TR-FRET-confirmed hits were validated in a GST-pull down assay using cell lysates of HEK293 cells co-expressing full-length constructs of both Venus-Flag-tagged CD44 and GST-tagged MSN. The Venus tag was used as a marker for CD44 protein expression. Dose responses were performed by the addition of 0.5 μl of compounds (in DMSO) to 100 μl of cell lysates which were diluted in 0.25% triton lysis buffer (150 mM NaCl, 10 mM HEPES, pH 7.5, 0.25% Triton X-100, phosphatase inhibitor (Sigma, P5726) and protease inhibitor cocktail (Sigma, P8340)). The mixtures were rotated for 1 h at 4 °C before the addition of 15 μL of 50% pre-equilibrated GST resin. After incubation on a rotator for 1.5 h at 4 °C, the GST resin was washed three times with 1 ml of 0.25% triton lysis buffer. SDS-sample buffer was added prior to boiling for 5 min. Samples were analysed by SDS-PAGE and immunoblotting, using anti-FLAG antibody for CD44 (1:1000 dilution of anti-Flag M2-HRP antibody; Sigma, A8592) and anti-GST antibody for GST-MSN (1:1000 dilution of anti-GST (2.6H1) Mouse mAb; Cell Signaling, 2624S). For GST detection, a secondary HRP-conjugated antibody was used (1:5000 dilution of AffiniPure Goat Anti-Mouse IgG (H+L); Jackson Immuno Research, 115–035-003).

Du Y., Bradshaw W.J., Leisner T.M., Annor-Gyamfi J.K., Qian K., Bashore F.M., Sikdar A., Nwogbo F.O., Ivanov A.A., Frye S.V., Gileadi O., Brennan P.E., Levey A.I., Axtman A.D., Pearce K.H., Fu H, & Katis V.L. (2023). Development of FERM domain protein-protein interaction inhibitors for MSN and CD44 as a potential therapeutic strategy for Alzheimer’s disease. bioRxiv.

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CD19BiTE Binding Assay

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To detect whether the secreted CD19BiTE could bind to the recombinant CD3ε protein (Cat# CDD-H52W1, Acro) and CD19 protein (Cat# CD9-H52H2, Acro), a FLAG-linked Elisa assay was performed. Briefly, 96-well plates were coated with CD3ε or CD19 protein at a concentration of 10 mg/ml. Then, supernatants of OVV-infected Hela-S3 cells were collected and added to the coated wells and incubated at 4 °C for 12 h. After that, the cell culture supernatant was removed, washed three times with 1 × TBS buffer, BiTE binding CD3ε and CD19 protein was determined by ELISA assays using anti-FLAG M2-HRP antibody (Cat#A8592, Sigma–Aldrich).
Binding assays were also performed with CD19⁺ tumor cells: K562/CD19, Raji, Farage, Pffeifer, Nalm-6 cells or CD3⁺ cells: Jurkat, CEM, Human T cells. Target cells (1 × 10⁵) were incubated on ice for one hour with the OVV-infected supernatants. BiTE binding was determined by flow cytometry using APC anti-DYKDDDDK Tag antibody (Cat# 637308, Biolegend, USA).

Lei W., Ye Q., Hao Y., Chen J., Huang Y., Yang L., Wang S, & Qian W. (2022). CD19-targeted BiTE expression by an oncolytic vaccinia virus significantly augments therapeutic efficacy against B-cell lymphoma. Blood Cancer Journal, 12(2), 35.

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Copper-Induced p4h Expression Assay

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Cells were grown under copper-depleted conditions to an OD₇₅₀ of 1.3 before p4h expression was induced by adding 1 μM CuSO₄ to the cultures. Samples were taken before induction (0 h) and after 1, 2, 4, 8, and 24 h. Samples were centrifuged (4 °C; 3000 g; 15 min) and re-suspended in ice-cold TBS buffer with 1 mM PMSF added. Cells were disrupted using glass beads (2 × 1 mm, 140 mg 0.4–0.6 mm, 120 mg 0.09–0.15 mm) and a Precellys Evolution homogenizer (Bertin Technologies) at 10,000 rpm for 4 × 30 s with a break of 30 s. The temperature of the samples was kept at 0–4 °C with a Cryolys Advanced Temperature Controller containing liquid nitrogen. Protein content was determined using a Bradford Dye Reagent Solution (ready-to-use, Alfa Aesar, Karlsruhe, Germany). A total amount of 7.5 μg protein was separated on 12% SDS-PAGE (Laemmli, 1970 (link)) and transferred to a nitrocellulose membrane (pore size 0.45 μm, NitroBind). Recombinant P4H protein was detected using an ANTI-FLAG M2-HRP antibody (Sigma-Aldrich, Cat. No. A8592) following the manufacturer’s protocol.

Brandenburg F., Theodosiou E., Bertelmann C., Grund M., Klähn S., Schmid A, & Krömer J.O. (2020). Trans-4-hydroxy-L-proline production by the cyanobacterium Synechocystis sp. PCC 6803. Metabolic Engineering Communications, 12, e00155.

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Stable Cell Line Generation via FLP/FRT

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All BioID stable cell lines were created with the FLP/FRT system and T-Rex 293T cells with constitutively expressed Tet repressor (Thermo Fisher Scientific). Stable cell lines expressing BioID-tagged genes of interest were generated by transfection with Lipofectamine 2000 (Thermo Fisher Scientific) and a combination of the appropriate pcDNA5/FRT/TO construct and Flipase expressing pOG44 plasmid. After recovery, cells were grown in DMEM containing 10% FBS, 1% PenStrep, and 50 µg/ml Hygromycin B. Gene expression was induced with 2 µg/ml doxycycline treatment for ~16 hr before cell harvest. Colonies were isolated, expanded, and screened for expression of the fusion proteins by western blotting with an anti-FLAG M2-HRP antibody (Sigma, mouse monoclonal, Cat #: A8592).

Christensen J.R., Kendrick A.A., Truong J.B., Aguilar-Maldonado A., Adani V., Dzieciatkowska M, & Reck-Peterson S.L. (2021). Cytoplasmic dynein-1 cargo diversity is mediated by the combinatorial assembly of FTS–Hook–FHIP complexes. eLife, 10, e74538.

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