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Flag antibody

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The Flag antibody is a laboratory reagent used for protein detection and purification. It is a monoclonal antibody that specifically binds to a short peptide sequence, known as the Flag tag, which can be genetically engineered onto target proteins. This allows for the identification and isolation of the tagged proteins from complex samples.

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Lab products found in correlation

Protein g dynabead, by Thermo Fisher Scientific (8 mentions) Mg132, by Merck Group (6 mentions) β actin, by Santa Cruz Biotechnology (5 mentions) Protein g magnetic beads, by Thermo Fisher Scientific (5 mentions) Flag peptide, by Merck Group (5 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (5 mentions) Protease inhibitor cocktail, by Merck Group (4 mentions) Gapdh, by Cell Signaling Technology (4 mentions) Bioruptor sonicator, by Diagenode (4 mentions) Ha antibody, by Merck Group (4 mentions) Protease inhibitor cocktail, by Roche (4 mentions) β actin, by Cell Signaling Technology (4 mentions) Lipofectamine 3000, by Thermo Fisher Scientific (4 mentions) Dynabeads, by Merck Group (3 mentions) Flag tag (flag), by Merck Group (3 mentions) Protein a g bead, by Santa Cruz Biotechnology (3 mentions) β actin antibody, by Merck Group (3 mentions) E cadherin, by Cell Signaling Technology (3 mentions) Protease inhibitor, by Roche (3 mentions) Ha antibody, by Santa Cruz Biotechnology (3 mentions)

199 protocols using flag antibody

1

GFP-p65 Cotransfection with RARα-Flag

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GFP-p65 or GFP-p65-S276A mutant was cotransfected with RARα-Flag to Calu-1 cells. RARα-Flag was stained with Flag antibody (MilliporeSigma, M2). The procedure of IF was as described previously (43 (link)).
Song H., Ye X., Liao Y., Zhang S., Xu D., Zhong S., Jing B., Wang T., Sun B., Xu J., Guo W., Li K., Hu M., Kuang Y., Ling J., Zhang T., Wu Y., Du J., Yao F., Chin Y.E., Wang Q., Zhou B.P, & Deng J. (2023). NF-κB represses retinoic acid receptor–mediated GPRC5A transactivation in lung epithelial cells to promote neoplasia. JCI Insight, 8(1), e153976.
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2

Epitope-tagged Protein Co-Immunoprecipitation

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Immunoprecipitation (IP) was performed by using the Dynabeads protein G IP kit (10007D, Thermo), following the manufacturer’s instruction. Briefly, HEK293T cells transfected with epitope-tagged expression plasmid(s) were lysed in the RIPA buffer (92590, Millipore) for 10 min at 4 ℃ and centrifuged at 16,000×g for supernatant collection. The Dynabeads were incubated with 4 μg HA antibody (sc-7392, Santa Cruz) or 2 μg FLAG antibody (F7425, MilliporeSigma) at room temperature for 10 min. Next, the Dynabeads were incubated with 500 µg cell lysate at 4 ℃ for 2 h. After washing, the IP samples were collected and used for Western blot (see next session) to complete the Co-IP assay. Appropriate host of IgG served as control which include mouse IgG (550878, BD, Franklin Lakes, NJ, USA) and rabbit IgG (550875, BD).
Tsai L.K., Peng M., Chang C.C., Wen L., Liu L., Liang X., Chen Y.E., Xu J, & Sung L.Y. (2023). ZSCAN4 interacts with PARP1 to promote DNA repair in mouse embryonic stem cells. Cell & Bioscience, 13, 193.
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3

Protein Extraction and Western Blotting

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As described previously (Sun et al., 2018), total proteins were extracted from infiltrated patches of N. benthamiana leaves using ×2 sodium dodecyl sulfate (SDS) sample buffer (100 mM Tris (pH 6.8), 4% SDS, 20% glycerol and 0.2% bromophenol blue) containing 10% β‐mercaptoethanol. Total yeast proteins were extracted as described (Kushnirov, 2000). Proteins were separated on 12.5% or 6% (for detection of 6Myc‐AtAGO1) polyacrylamide gels, and transferred onto polyvinylidene fluoride membranes. The membranes were blotted with the FLAG antibody (Sigma‐Aldrich), c‐Myc antibody (Sigma‐Aldrich), or polyclonal antiserum against GFP or NbSKP1, and subsequently detected by goat anti‐rabbit horseradish peroxidase‐conjugated antibody (Bio‐Rad) followed by chemiluminescence detection (GE Healthcare). To quantify the protein, coomassie brilliant blue R250 was used (0.1% in 50% methanol : 12% acetic acid) to stain the gel overnight with gentle shaking.
Li Y., Sun Q., Zhao T., Xiang H., Zhang X., Wu Z., Zhou C., Zhang X., Wang Y., Zhang Y., Wang X., Li D., Yu J., Dinesh‐Kumar S.P, & Han C. (2019). Interaction between Brassica yellows virus silencing suppressor P0 and plant SKP1 facilitates stability of P0 in vivo against degradation by proteasome and autophagy pathways. The New Phytologist, 222(3), 1458-1473.
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4

Affinity Purification of FLAG-Tagged Proteins

Cited 2 times
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The IP and affinity purification were performed as described previously with minor modifications (38 (link)). Briefly, ∼2 g of flower tissue collected from SAC3B-3XFlag transgenic T4 plants were ground to a fine powder in liquid nitrogen and suspended in 15 ml of lysis buffer (50 mM Tris [pH 7.6], 150 mM NaCl, 5 mM MgCl2, 10% glycerol, 0.1% NP-40, 0.5 mM DTT, 1 mg/ml pepstatin, 1 mM PMSF and 1% protease inhibitor cocktail [Sigma, P9599]). The tissue was further homogenized by douncing and then centrifuged at 4°C for 25 min at 12 500 rpm. Eighty microliters of Dynabeads (Invitrogen, 10003D) that had been conjugated with Flag antibody (Sigma, F1804) according to the manufacturer's instructions were added to the supernatant for IP. After incubation at 4°C with rotation for 2.5 h, the Flag beads were washed once with 5 ml of LB for 15 min, and then 5 times for 5 min with 1 ml of LB, and twice for 5 min with 1 ml PBS. The protein was then subjected to Mass Spectrometry analysis according to previously described procedures (15 (link)).
Yang Y., La H., Tang K., Miki D., Yang L., Wang B., Duan C.G., Nie W., Wang X., Wang S., Pan Y., Tran E.J., An L., Zhang H, & Zhu J.K. (2016). SAC3B, a central component of the mRNA export complex TREX-2, is required for prevention of epigenetic gene silencing in Arabidopsis. Nucleic Acids Research, 45(1), 181-197.
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5

ChIP Assay for DCP2 Protein

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Chromatin immunoprecipitation (ChIP) was carried out according to the manufacturer's protocol. First, after crosslinked with 1% formalin, cells were seeded in SDS buffer. Nuclei were pelleted and resuspended in IP buffer (2 volumes SDS lysis buffer:1 volume Triton-X buffer (100 mM Tris-Cl, pH 8.6, 100 mM NaCl, 5 mM EDTA pH 8.0, 5% Triton X-100)). The lysates were sonicated using Bioruptor sonicator for 12 cycles of 30 s and centrifuged at maximum speed. ChIP for decapping enzyme homolog (DCP2) was carried out using a Flag antibody (Sigma, SAB4301135). Eluted DNA fragments were further purified using Minelute PCR Purification Kit (Qiagen) and then amplified by qPCR. The primers are listed in Table S1.
Dabin R., Wei C., Liang S., Ke C., Zhihan W, & Ping Z. (2022). Astrocytic IGF-1 and IGF-1R Orchestrate Mitophagy in Traumatic Brain Injury via Exosomal miR-let-7e. Oxidative Medicine and Cellular Longevity, 2022, 3504279.
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6

SLX4IP Protein Interaction Analysis

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RNA was isolated, quantified, and analyzed as described previously.35 (link) Protein isolation, western blotting analysis and quantification was completed as published previously.35 (link) Primary antibody dilutions are as follows: 1:10,000 GAPDH (Cell Signaling, 2118), 1:3,000 SLX4IP (Sigma, HPA046372), 1:3,000 FLAG (Sigma, F1804), 1:5,000 TRF2 (Novus Biologicals, NB110–57130), 1:1,000 PML (Cell Signaling, E5R8T and Santa Cruz Biotechnology, sc-5621), 1:3,000 p21 (Cell Signaling, 2947).
Coimmunoprecipitation experiments were carried out using the Dynabeads™ Co-Immunoprecipitation Kit according to manufacturer instructions (ThermoFisher Scientific). The provided immunoprecipitation (IP) buffer included was modified with an additional 50 mM NaCl and 0.05% Triton X-100. Cells were grown in 15 cm dishes and scraped using the modified IP buffer, treated with 5 μL of recombinant DNaseI (Sigma), rotated at 4° C for one hour, and cleared by centrifugation. Following protein quantification, 2 mg of protein was incubated with 1.5 mg of Dynabeads coupled with 10 µL of FLAG antibody (Sigma, F1804) or 20 µL of SLX4IP antibody (Sigma, HPA046372) per protocol. After completion, whole cell lysates and coimmunoprecipitation samples were analyzed by western blotting as described above.
Mangosh T.L., Grabowska M.M, & Taylor D.J. (2021). SLX4IP N-terminus dictates telomeric localization in ALT-like castration-resistant prostate cancer cell lines. The Prostate, 81(15), 1235-1251.
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7

Western Blot Analysis of Cell Signaling

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Twenty μg of protein per sample were loaded in SDS-PAGE in 10% (for E2F1,Chk1 and p53) or 8% (for Rb or PARP-1) polyacrylamide gels, and then transferred onto nitrocellulose membranes. Antibody dilutions were as follows: Chk1- 1:500 (sc-377231), E2F1- 1:1000 (sc-193), Rb- 1:200 (sc-102), PARP-1- 1:1000 (H-300: sc-25780), Cleaved Caspase-3 (Asp175) Antibody #9661 1:1000, p53 Antibody #9282 1:1000 (Cell signaling) in 5% fat free milk 0’05% TTBS. HA Antibody- 1:2000 (Boehringer mannheim), Flag antibody- 1:2000 (Sigma)
Bargiela-Iparraguirre J., Prado-Marchal L., Fernandez-Fuente M., Gutierrez-González A., Moreno-Rubio J., Muñoz-Fernandez M., Sereno M., Sanchez-Prieto R., Perona R, & Sanchez-Perez I. (2016). CHK1 expression in Gastric Cancer is modulated by p53 and RB1/E2F1: implications in chemo/radiotherapy response. Scientific Reports, 6, 21519.
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8

Protein Expression Analysis in Stem Cells

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Western blot analysis and co-immunoprecipitation was performed as previously [16] (link) with antibodies indicated, CK2α (Santa Cruz, sc-6479), CK2α′ (Santa Cruz, sc-6481), Nanog (Cell Signaling, 4903), Oct4 (Cell Signaling, 4286), Sox2 (Cell Signaling, 2748), beta-actin (Cell Signaling, 4967), TAp73 (IMGENEX,IMG-246), p73 (IMGENEX,IMG-259A), Oct-1 (Santa Cruz, sc-53830), Flag antibody(Sigma, M2), PUMA (Cell Signaling, 4976).
Lu H., Yan C., Quan X.X., Yang X., Zhang J., Bian Y., Chen Z, & Van Waes C. (2014). CK2 Phosphorylates and Inhibits TAp73 Tumor Suppressor Function to Promote Expression of Cancer Stem Cell Genes and Phenotype in Head and Neck Cancer. Neoplasia (New York, N.Y.), 16(10), 789-800.
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9

Immunoprecipitation and Western Blot

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Cells were transfected as indicated, and then were collected by immunoprecipitation lysis buffer (Beyotime, Shanghai, China). Next equal amounts of cell lysis were incubated with FLAG antibody (1:200; Sigma) immobilised onto Protein G-Sepharose beads for 6 hours at 4 °C with gentle rotation. Then the beads were washed by lysis buffer three times. After adding with SDS-PAGE sample loading buffer and subsequently boiling, the beads were centrifuged to acquire supernatant for western blot.
Song S., Liu B., Zeng X., Wu Y., Chen H., Wu H., Gu J., Gao X., Ruan Y, & Wang H. (2022). Reticulon 2 promotes gastric cancer metastasis via activating endoplasmic reticulum Ca2+ efflux-mediated ERK signalling. Cell Death & Disease, 13(4), 349.
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10

Subcellular Localization of Ubi-MCS-3FLAG-SV40-EGFP

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Ubi-MCS-3FLAG-SV40-EGFP was expressed in HEK293 cells, and immunofluorescence confocal microscopy was employed to determine its subcellular localization using GM130 antibody (BD Bioscience, San Jose, CA, USA) and anti-mouse-cy3 (Jackson ImmunoResearch, West Grove, PA, USA) for the Golgi apparatus, Flag antibody (Sigma, CA, USA) and anti-rabbit-647 (Jackson ImmunoResearch) for βCGRP, and calnexin antibody (Sigma) and anti-rabbit-647 for the ER.
Liu Q.C., Chen F., Wu C.Y., Gao F., Zhuang Z.H., Chen J.T., Cai B., Zhang T., Guo L., Lin L.Q., Zhao C.F, & Lin X.H. (2017). CALCB splice region pathogenic variants leading to plasma cell neurotropic enrichment in type 1 autoimmune pancreatitis. Cell Death & Disease, 8(2), e2591-.
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