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Anti flag f7425

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Anti-Flag (F7425) is a laboratory reagent produced by Merck Group. It is a monoclonal antibody that specifically binds to the FLAG epitope tag, which is commonly used for the detection and purification of recombinant proteins. The core function of this product is to facilitate the identification and isolation of FLAG-tagged proteins in various experimental settings.

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Lab products found in correlation

Lt1 transfection reagent, by Mirus Bio (2 mentions) Donkey anti mouse 488, by Jackson ImmunoResearch (2 mentions) Mouse monoclonal anti p65 clone f 6, by Santa Cruz Biotechnology (2 mentions) Goat anti rabbit 546, by Jackson ImmunoResearch (2 mentions) Anti p53 fl393, by Santa Cruz Biotechnology (2 mentions) Anti c myc c3956, by Merck Group (2 mentions) Ab2851, by Abcam (2 mentions) Ab9110, by Abcam (2 mentions) Anti gadd45a sc 797, by Santa Cruz Biotechnology (2 mentions) Gtx124207, by GeneTex (2 mentions) Rnase h1, by Thermo Fisher Scientific (2 mentions) Nb300 516, by Novus Biologicals (2 mentions) Anti β actin a5316, by Merck Group (1 mentions) Anti cleaved caspase 3 d175, by Cell Signaling Technology (1 mentions) Anti ha sc 7392, by Santa Cruz Biotechnology (1 mentions) Protein g sepharose 4 fast flow, by GE Healthcare (1 mentions) Kod plus mutagenesis kit, by Toyobo (1 mentions) Anti ha clone 3f10, by Roche (1 mentions) 3xflag peptide, by Merck Group (1 mentions) Mg132 hy 13259, by MedChemExpress (1 mentions)

35 protocols using anti flag f7425

1

Visualizing NF-κB Translocation in HeLa Cells

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HeLa cells were transfected with EV or plasmids encoding N-terminal TAP-tagged B14, C2, C2-B (aa 1−212), C2-K (aa 213−512) or F3 using the LT1 transfection reagent (MirusBio). After 24 h cells were starved in DMEM without serum for 3 h and then stimulated with 40 ng ml−1 TNFα for 30 min. Cells were washed three times in ice-cold PBS and processed for immunofluorescence staining and imaging as described [47 (link)]. Polyclonal rabbit anti-Flag (F7425, Sigma-Aldrich) and mouse monoclonal anti-p65 clone F-6 (sc-8008; Santa Cruz) were used as the primary antibodies and goat anti-rabbit 546 and donkey anti-mouse 488 were used as the secondary antibodies (Jackson Immunoresearch). Images were analysed using the Zeiss Zen microscope software and ImageJ. Experiments were performed in triplicate and carried out three times. For each repeat, 100 Flag-positive cells were analysed for each condition and the percentage of cells showing nuclear p65 determined.
Zhang R.Y., Pallett M.A., French J., Ren H, & Smith G.L. (2022). Vaccinia virus BTB-Kelch proteins C2 and F3 inhibit NF-κB activation. The Journal of general virology, 103(10), 10.1099/jgv.0.001786.
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2

Western Blot Analysis of Protein Levels

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Standard western blot assays were used to analyze the levels of protein [14 , 37 (link)]. Anti-p53 (FL393, 1:2 000 dilution, Santa Cruz), anti-MDM2 (2A10, 1:1 000), anti-Flag (F7425,1:10 000 dilution, Sigma), anti-BAG5(ARP61996-P050, 1:1 000 dilution, Aviva Systems Biology, San Diego, CA, USA), anti-HA (sc-7392, 1:2 000 dilution, Santa Cruz), anti-CHIP (sc-66830, 1:1 000 dilution, Santa Cruz), anti-cleaved-Caspase 3 (D175, 1:1 000 dilution, Cell Signaling, Danvers, MA, USA) and anti-β-actin(A5316, 1:20 000 dilution, Sigma) antibodies were used to determine the levels of p53, MDM2, Flag-BAG5, BAG5, HA-BAG5, CHIP, cleaved caspase 3 and β-actin, respectively.
Yue X., Zhao Y., Huang G., Li J., Zhu J., Feng Z, & Hu W. (2016). A novel mutant p53 binding partner BAG5 stabilizes mutant p53 and promotes mutant p53 GOFs in tumorigenesis. Cell Discovery, 2, 16039-.
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3

Signaling Pathway Activation Assay

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The following chemical reagents and antibodies were used in this study: anti-TBK1 (3504), anti-phospho-TBK1 (5483), anti-phospho-IRF3 (29047), anti-phospho-IRF7 (24129), anti-phospho-p65 (3033), anti-phospho-p38 (9215), anti-phospho-Erk1/2 (9101), anti-STING (13647), anti-cGAS (15102), anti-RIG-I (4200), anti-phospho-STING (72971), and anti-β-arrestin 2 (3857 S; all from Cell Signaling Technology); anti-β-arrestin 2 (ab54790; from Abcam) are diluted in the ratio of 1:1000; anti-Flag M2 Affinity Gel (A2220), anti-HA (H9658), anti-HA (H6908), and anti-Flag (F7425; all from Sigma-Aldrich) are diluted in the ratio of 1:10000; anti-GST (CW0085M) and anti-His (CW0083M; both from Cwbio) are diluted in the ratio of 1:10,000; and Protein G Sepharose 4 Fast Flow (GE Healthcare). Expression constructs for Flag-RIG-I, Flag-RIG-I-N, Flag-TBK1, Flag-IRF3, Flag-cGAS, Flag-TRAF3, HA-cGAS, HA-CK1ɛ, and HA-Ubs were obtained from Dr. B. Ge (Tongji University, Shanghai, China), and Flag-STING was obtained from Dr. B. Sun (Shanghai Institute of Biochemistry and Cell Biology, Shanghai, China). Site-directed point mutagenesis of β-arrestin 2 was performed using the KOD Plus Mutagenesis Kit (SMK101, TOYOBO) according to the manufacturer’s instructions.
Zhang Y., Li M., Li L., Qian G., Wang Y., Chen Z., Liu J., Fang C., Huang F., Guo D., Zou Q., Chu Y, & Yan D. (2020). β-arrestin 2 as an activator of cGAS-STING signaling and target of viral immune evasion. Nature Communications, 11, 6000.
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4

Immunoaffinity Purification of Hrd1 Complex

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Approximately 30 OD600 of cells were harvested and resuspended in IPB (25mM HEPES pH 7.4, 200mM NaCl) supplemented with a protease inhibitor cocktail. Cells were lysed using glass beads and cell debris were removed by centrifugation at 6,000 g for 1 min. Membrane fractions were collected by ultra-centrifugation at 42,000 rpm for 20 mins. Membranes were homogenized and solubilized in IPB containing 1% Nonidet P-40 for 1 hr. The extract was then incubated with 7 ul of anti-FLAG M2 resin for 2 hrs. The beads were washed three times with IPB containing 0.1% Nonidet P-40 and bound proteins were eluted with buffer containing 0.2 mg/ml of 3xFLAG peptide (Sigma). Eluted proteins were subjected to SDS-PAGE and immunoblotting. HA- and FLAG- tagged Hrd1 were detected using anti-HA (clone 3F10, Roche) and anti-FLAG (F7425, Sigma) antibodies, respectively.
Wu X., Siggel M., Ovchinnikov S., Mi W., Svetlov V., Nudler E., Liao M., Hummer G, & Rapoport T.A. (2020). Structural basis of ER-associated protein degradation mediated by the Hrd1 ubiquitin ligase complex. Science (New York, N.Y.), 368(6489), eaaz2449.
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5

Visualizing NF-κB Translocation in HeLa Cells

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HeLa cells were transfected with EV or plasmids encoding N-terminal TAP-tagged B14, C2, C2-B (aa 1−212), C2-K (aa 213−512) or F3 using the LT1 transfection reagent (MirusBio). After 24 h cells were starved in DMEM without serum for 3 h and then stimulated with 40 ng ml−1 TNFα for 30 min. Cells were washed three times in ice-cold PBS and processed for immunofluorescence staining and imaging as described [47 (link)]. Polyclonal rabbit anti-Flag (F7425, Sigma-Aldrich) and mouse monoclonal anti-p65 clone F-6 (sc-8008; Santa Cruz) were used as the primary antibodies and goat anti-rabbit 546 and donkey anti-mouse 488 were used as the secondary antibodies (Jackson Immunoresearch). Images were analysed using the Zeiss Zen microscope software and ImageJ. Experiments were performed in triplicate and carried out three times. For each repeat, 100 Flag-positive cells were analysed for each condition and the percentage of cells showing nuclear p65 determined.
Zhang R.Y., Pallett M.A., French J., Ren H, & Smith G.L. (2022). Vaccinia virus BTB-Kelch proteins C2 and F3 inhibit NF-κB activation. The Journal of general virology, 103(10), 10.1099/jgv.0.001786.
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6

Investigating Ferroptosis Regulators in Cells

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Anti-MFG-E8 (sc-271574; 1:200 dilution for immunoblotting, immunofluorescence, and immunohistochemistry; 2 µg per 1 mg of total protein for immunoprecipitation), anti-USP14 (sc-398009, 1:200 for immunoblotting, 2 µg per 1 mg of total protein for immunoprecipitation), and anti-GPx4 (sc-166570, 1:400 for immunoblotting) antibodies were purchased from Santa Cruz Biotechnology, USA. Anti-SLC7A11 (12691, 1:1000 for immunoblotting) and anti-Ub (3936, 1:1000) were from Cell Signaling Technology, USA. Anti-Flag (F7425, 1:1000) was from Sigma-Aldrich, USA. Antibodies against β-actin (60008–1-Ig, 1:5000), β-tubulin (10068-1-AP, 1:1000), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (60004-1-Ig, 1:1000) were from Proteintech, China. Anti-USP14 (AF301319, 1:100 for immunofluorescence) was from AiFang Biological, China. Anti-GPx4 (ab125066, Abcam, UK, 1:100), and anti-SLC7A11 (26864-1-AP, Proteintech, China, 1:50) antibodies were used for immunohistochemistry (IHC) staining. Immobilized protein A/G beads (45350) were purchased from Thermo Fisher Scientific, USA. Control IgG (sc-2025) came from Santa Cruz Biotechnology, USA. Cycloheximide (CHX) (S7418), RSL3 (S8155), and Ferrostatin-1 (Fer-1) (S7243) were from Selleck, China. MG132 (HY-13259) was purchased from MedChemExpress, USA.
Cui Y., Luo L., Zeng Z., Liu X., Li T., He X., Ma Y., Meng W., Zeng H., Long Y., Zong D, & Chen Y. (2023). MFG-E8 stabilized by deubiquitinase USP14 suppresses cigarette smoke-induced ferroptosis in bronchial epithelial cells. Cell Death & Disease, 14(1), 2.
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7

Co-Immunoprecipitation of Chondrocyte Transcription Factors

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We collected whole cell lysates from human articular chondrocytes using an M-PER kit, and performed co-IP using a Catch and Release kit (Upstate Biotechnology, Lake Placid, NY) with anti-Flag (F7425, Sigma-Aldrich, St. Louis, MO), anti-Runx1 (23980), anti-Sox5 (94396), anti-Sox6 (30455) and anti-Sox9 (185230) antibodies (Abcam, Cambridge, UK). Immune complexes were eluted and subjected to SDS-PAGE. Mammalian two-hybrid assays were performed with the Checkmate Mammalian Two-Hybrid System (Promega, Madison, WI).
Yano F., Ohba S., Murahashi Y., Tanaka S., Saito T, & Chung U.I. (2019). Runx1 contributes to articular cartilage maintenance by enhancement of cartilage matrix production and suppression of hypertrophic differentiation. Scientific Reports, 9, 7666.
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8

Characterization of TRPM4 and Neuronal Markers

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Two different rabbit polyclonal antibodies against TRPM4, ACC-044 (RRID:AB_2040250) and ab104572 (RRID:AB_10712148), were obtained from Alomone (Israel) and Abcam (United States) respectively. Monoclonal anti-TRPM4 L88/86 hybridoma tissue culture supernatant (RRID: AB_2716758). Monoclonal anti-TRPM4 clone 10H5 (RRID: AB_2208624) was obtained from Origene (United States). Mouse monoclonal and rabbit polyclonal anti-MAP2 antibodies (ab11267, RRID:AB_297885; and ab32454">ab32454, RRID:AB_776174, respectively) were obtained from Abcam (United States). Anti-NeuN (MAB377, RRID:AB_2298772), anti-GAD67 (MAB5406, RRID:AB_2278725) mouse monoclonal antibodies, and the anti-Neurogranin (AB5620, RRID:AB_2171427) rabbit polyclonal antibody were obtained from Millipore (United States). Anti-AnkG (75-146, RRID:AB_10673030) mouse monoclonal antibody N106/36 was obtained from the UC Davis/NIH NeuroMab Facility (United States), and rabbit polyclonal anti-FLAG (F7425, RRID:AB_439687) was obtained from Sigma (United States) (Table 1).
Riquelme D., Silva I., Philp A.M., Huidobro-Toro J.P., Cerda O., Trimmer J.S, & Leiva-Salcedo E. (2018). Subcellular Localization and Activity of TRPM4 in Medial Prefrontal Cortex Layer 2/3. Frontiers in Cellular Neuroscience, 12, 12.
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9

Western Blot Protein Quantification

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Western blot was performed as in Neves et al. (2012 (link)). Protein samples were separated in 12% polyacrylamide gels, membranes incubated overnight with anti-FLAG (F7425, Sigma, 1:5000) or anti-GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA, USA 1:15,000). Secondary antibodies goat anti-rabbit (P0488, Dako, 1:2000) and donkey anti-mouse (715-036-150, Jackson/Affinipure, 1:2000) coupled to horseradish peroxidase were incubated for 1 h at room temperature. Optical density of immunoreactive bands was quantified on a ChemiDoc XRS System and Quantity One Software v4.6.3 (Bio-Rad). Values were normalized to GAPDH.
Gálvez H., Tena J.J., Giraldez F, & Abelló G. (2017). The Repression of Atoh1 by Neurogenin1 during Inner Ear Development. Frontiers in Molecular Neuroscience, 10, 321.
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10

Immunoblotting Antibody Detection Protocol

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Regular immunoblotting was performed for protein detection following SDS‐PAGE electrophoresis. The primary antibodies used for western blot analysis include: anti‐FLAG® (F7425) (1:2000), anti‐FLAG® M2 (F1804) (1:4000), anti‐c‐Myc (C3956) (1:2000) from Sigma‐Aldrich (St. Louis, MO); anti‐Myc Tag, clone 4A6 (05‐724) (1:4000) from Millipore Corporation (Billerica, MA); anti‐ CK1α1 [EPR1961(Mendoza et al. 2007)] (ab108296) (1:2000), anti‐CK1δ [AF12G4] (ab85320) (1:4000), anti‐CK1ε [AF6C1] (ab82426) (1:2000) from abcam® (Cambridge, MA); anti‐HaloTag® (G9211) (1:1000) from Promega Corporation (Madison, WI). Two secondary antibodies were used: ECL anti‐rabbit IgG (NA934V; GE Healthcare; Little Chalfont, U.K.) and anti‐mouse IgG+IgM HRP (ab47827; abcam®; Cambridge, MA).
Wang S., Hu Y., Yang J., Smith C.E., Richardson A.S., Yamakoshi Y., Lee Y., Seymen F., Koruyucu M., Gencay K., Lee M., Choi M., Kim J., Hu J.C, & Simmer J.P. (2015). Fam83h null mice support a neomorphic mechanism for human ADHCAI. Molecular Genetics & Genomic Medicine, 4(1), 46-67.
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