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Anti flag

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Sourced in Japan, United States

Anti-FLAG is a laboratory reagent used to detect and purify proteins that have been engineered to express a specific peptide tag, known as the FLAG tag. The Anti-FLAG reagent binds to the FLAG tag, allowing for the identification and isolation of the tagged protein from a complex mixture.

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Lab products found in correlation

Anti ha, by Medical & Biological Laboratories (2 mentions) Lsm 710, by Zeiss (2 mentions) Paraformaldehyde (pfa), by Merck Group (2 mentions) Gsno donor, by Merck Group (2 mentions) Proteostat dye, by Medical & Biological Laboratories (2 mentions) Anti myc, by Medical & Biological Laboratories (2 mentions) Ab109012, by Abcam (1 mentions) Anti ubiquitin, by Proteintech (1 mentions) Anti flag, by Proteintech (1 mentions) Anti slc7a11, by Novus Biologicals (1 mentions) Anti slc7a11, by Proteintech (1 mentions) Nutlin 3, by Selleck Chemicals (1 mentions) Anti gapdh, by Cell Signaling Technology (1 mentions) Ab80588, by Abcam (1 mentions) Anti β actin, by Cell Signaling Technology (1 mentions) Ab32049, by Abcam (1 mentions) Ab68477, by Abcam (1 mentions) Erastin, by Selleck Chemicals (1 mentions) Anti p53, by Proteintech (1 mentions) Anti nrf2, by Proteintech (1 mentions)

Close spelling variants of this product

Anti-Flag antibody (4 citations) Rabbit anti-FLAG (3 citations) Mouse anti-Flag monoclonal antibody (2 citations) Anti-Flag and anti-Myc antibodies (2 citations)

11 protocols using anti flag

1

Western Blot Analysis of Cellular Proteins

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The antibodies included the following: anti-SQSTM1/p62 (M162-3, Medical Biological Laboratories, Japan), anti-SQSTM1/p62 (ab109012, Abcam, USA), anti-p53 (sc-126, Santa Cruze Biotechnology, USA), anti-p53 (10,442–1-AP, Proteintech, China), anti-mutant p53 (ab32049, Abcam), anti-NRF2 (M200-3, Medical Biological Laboratories), anti-NRF2 (16,396–1-AP, Proteintech), anti-ubiquitin (10,201–2-AP, Proteintech), anti-GAPDH (#5174, Cell Signaling Technology), anti-β-actin (#4970, Cell Signaling Technology), anti-HA (M180-3 and M561, Medical Biological Laboratories), anti-His (D291-3, Medical Biological Laboratories), anti-Flag (M185, Medical Biological Laboratories), anti-Flag (20,543–1-AP, Proteintech), anti-SLC7A11 (NB300-318, Novus, USA), anti-SLC7A11 (26,864–1-AP, Proteintech) anti-HO1 (ab68477, Abcam), anti-HO1 (10,701–1-AP, Proteintech), anti-NQO1 (ab80588, Abcam), anti-Keap1 (10,503–2-AP, Proteintech).
The reagents included the following: Erastin (S7242, Selleck, USA), APR-246 (HY-19980, MCE, USA), Pifithrin-α (HY-15484, MCE), Nutlin-3 (S1061, Selleck).
Yuan F., Sun Q., Zhang S., Ye L., Xu Y., Deng G., Xu Z., Zhang S., Liu B, & Chen Q. (2022). The dual role of p62 in ferroptosis of glioblastoma according to p53 status. Cell & Bioscience, 12, 20.
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2

Immunostaining of MEF Cells with GSNO

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For immunostaining, MEF cells were cultured in 24 wells plate with coverslips. After cell seeding for 24 hours, the cells were further treated with GSNO donor (Sigma Aldrich, 10uM) for 24 hours. Cells were fixed with 4% paraformaldehyde (Sigma Aldrich), and blocked with a blocking solution (normal goat serum) accompanied with 0.1% saponin for permeabilization. After blocking, the cells were incubated in 0.1% saponin with primary antibody: anti-FLAG (Medical & Biological Laboratories), secondary antibody and fluorescence-probe: Alex Flour 633 goat anti-mouse, ProteoStat Dye (1:1000), DAPI (1ug/ml). Images were taken in multi-tracking mode on a laser scanning confocal microscope (LSM710, Carl Zeiss) with a 63 plan apochromat 1.4 NA objective.
Yi W., Zhang Y., Liu B., Zhou Y., Liao D., Qiao X., Gao D., Xie T., Yao Q., Zhang Y., Qiu Y., Huang G., Chen Z., Chen C, & Ju Z. (2021). Protein S-nitrosylation regulates proteostasis and viability of hematopoietic stem cell during regeneration. Cell reports, 34(13), 108922.
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3

Separation and Analysis of Protein Complexes

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Protein complexes of anthers or crude mitochondria were separated by Blue Native polyacrylamide gel electrophoresis (BN-PAGE) according to [21 (link)]. A NativePAGE Novex BisTris Gel system (Thermo Fisher Scientific, Waltham, MA, USA) was used. Separated complexes were blotted onto a Hybond-P PVDF membrane (GE Healthcare, Little Chalfont, UK) according to the manufacturer’s instruction manual. Primary antisera were anti-preSATP6 [10 (link)], anti-COXI [10 (link)], and anti-FLAG (Medical and Biological Laboratories, Nagoya, Japan). Antisera were diluted as described in [25 (link)]. The secondary antibody was HRP-conjugated goat anti-mouse IgG and HRP-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA, USA). Conditions for quantification were modified as previously described [25 (link)]. Uncropped images are provided in Additional file 8.
Arakawa T., Matsunaga M., Matsui K., Itoh K., Kuroda Y., Matsuhira H., Kitazaki K, & Kubo T. (2020). The molecular basis for allelic differences suggests Restorer-of-fertility 1 is a complex locus in sugar beet (Beta vulgaris L.). BMC Plant Biology, 20, 503.
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4

Immunoblotting Analysis of Protein Lysates

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The primary tissue lysates and whole cell lysates were prepared for immunoblotting as described previously 25 (link). Equal amounts of total proteins (30 µg) were subjected to SDS-PAGE and immunoblotting analysis with specific antibodies. The primary antibody against Cyclin D1 was purchased from Cell Signaling Technology (Danvers, MA). Anti-USP5 antibody was obtained from Proteintech Group (Wuhan, China). Anti-TUFM and anti-EBF1 antibodies were purchased from ABclonal Biotechnology (Hubei, China). Anti-Flag, Myc and HA antibodies were purchased from Medical & Biological Laboratories (Tokyo, Japan). Anti-GAPDH antibody was purchased from Abgent Biotechnology (Suzhou, China). Horseradish peroxidase-conjugated anti-mouse and anti-rabbit IgG antibodies were purchased from Beyotime Biotechnology (Nantong, China).
Xu X., Huang A., Cui X., Han K., Hou X., Wang Q., Cui L, & Yang Y. (2019). Ubiquitin specific peptidase 5 regulates colorectal cancer cell growth by stabilizing Tu translation elongation factor. Theranostics, 9(14), 4208-4220.
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5

ChIP Assay for Protein-DNA Interactions

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The ChIP kit (Millipore, Burlington, MA, USA, 17-295) was employed according to the manufacturer’s instructions and the ChIP assay was performed as described previously25 (link). Briefly, cells (1 × 107 per assay) were bathed in 1% formaldehyde at 25°C for 10 min for cross-linking of proteins and DNA and then lysed in sodium dodecyl sulfate buffer containing protease inhibitors. DNA was sheared by sonication using a Sonic Dismembrator Model 500 (Fisher Scientific, Pittsburgh, PA, USA). Immunoprecipitation was carried out by incubating with 1 μg of anti-FLAG (Medical & Biological Laboratories, M185-3L), anti-NANOG (Bethyl Laboratories, A300-379A) antibodies or rabbit IgG (Millipore, Billerica, MA, USA, PP64) for 16 h and then the immunoprecipitated DNA was quantified by real-time qPCR using the following primer set: 5′-GCAGGACTTGAGAAGCCTCTC-3′ (forward) and 5′-GACGCAGCTGCACACATG-3′ (reverse). Each sample was assayed in triplicate, and the amount of precipitated DNA was calculated as the percentage of the input sample.
Oh S.J., Lim J.Y., Son M.K., Ahn J.H., Song K.H., Lee H.J., Kim S., Cho E.H., Chung J.Y., Cho H., Kim H., Kim J.H., Park J., Choi J., Hwang S.W, & Kim T.W. (2023). TRPV1 inhibition overcomes cisplatin resistance by blocking autophagy-mediated hyperactivation of EGFR signaling pathway. Nature Communications, 14, 2691.
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6

Co-immunoprecipitation Assay for TaMOR-GFP and ARF5-Flag

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The coding sequence of TaMOR was cloned into vector pCAMBIA1300‐GFP to form TaMOR‐GFP. The coding sequence of ARF5 was cloned into vector to pCAMBIA1300‐Flag to form ARF5‐Flag. They were introduced into Agrobacterium tumefaciens strain EHA105 and infiltrated into N. benthamiana leaves. After 48 h, the infiltrated leaves were ground to powder in liquid nitrogen and resuspended in 500 μl protein extraction buffer. The Co‐IP assay was performed as previously described (Cai et al., 2021 (link)). Anti‐GFP mAb magnetic beads (Medical Biological Laboratories, D153‐11), Anti‐GFP (Medical Biological Laboratories, 598‐7) and anti‐Flag (Medical Biological Laboratories, M185‐7) antibodies were used in the assay.
Li C., Wang J., Li L., Li J., Zhuang M., Li B., Li Q., Huang J., Du Y., Wang J., Fan Z., Mao X, & Jing R. (2021). TaMOR is essential for root initiation and improvement of root system architecture in wheat. Plant Biotechnology Journal, 20(5), 862-875.
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7

Quantification of Vif and A3G Proteins in HIV-1 Virions and Cells

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Western blotting analysis was performed as described previously [37 (link),38 (link)]. Briefly, pro-viral clones or pcNLmini-RI-based clones were transfected into HEK 293T cells by Lipofectamine2000, and unless otherwise noted, on day 1, post-transfection cell lysates were prepared with 1 × TNE buffer. Analyses of Vif and A3G expression levels in virions and cells were carried out similarly as described previously [43 (link),44 (link)]. Briefly, HEK 293T cells were co-transfected with pro-viral clones along with a FLAG-tagged A3G expression vector by the calcium phosphate coprecipitation method. On day 1 post-transfection, virions were collected by ultracentrifugation, and the lysates of virions and cells were prepared with 1 × TNE buffer. Anti-HIV-1 Vif 319 (catalog no. ab66643; Abcam, Tokyo, Japan), anti-Gag-p24 (#3537, NIH Research and References Reagent Program), anti-FLAG (FLA-1, MEDICAL & BIOLOGICAL LABORATORIES), and anti-β-actin clone AC-15 (Sigma-Aldrich, Burlington, MA, USA) antibodies were used for immunoblotting analyses.
Koma T., Doi N., Takemoto M., Watanabe K., Yamamoto H., Nakashima S., Adachi A, & Nomaguchi M. (2021). The Expression Level of HIV-1 Vif Is Optimized by Nucleotide Changes in the Genomic SA1D2prox Region during the Viral Adaptation Process. Viruses, 13(10), 2079.
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8

Comprehensive Biochemical Assay Protocol

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DMSO was purchased from Servicebio (GC203005). The AKT inhibitor SC66 was obtained from MedChemExpress (HY-19832). TMZ was purchased from Selleck (S1237).
The following antibodies were used: anti-RND1 (GTX83709, GeneTex), anti-GAPDH (#5174, Cell Signalling Technology, CST), anti-Flag (M185, Medical Biological Laboratories), anti-E-cadherin (20874–1-AP, Proteintech), anti-N-cadherin (22018–1-AP, Proteintech), anti-Vimentin (10366–1-AP, Proteintech), anti-Snail1 (sc -28,199, Santa Cruz), anti-mmp2 (10373–2-AP, Proteintech), anti-AKT (GTX121937, GeneTex), anti-Phospho-AKT (#4060, CST), anti-GSK-3β (#12456, CST), anti-Phospho-GSK-3β (#9323, CST), anti-β-catenin (51067–2-AP, Proteintech), anti-MAPK (#4511, CST), anti-Phospho-MAPK (#9212, CST) and anti-Smad3 (66516–1-Ig, Proteintech).
Sun Q., Xu J., Yuan F., Liu Y., Chen Q., Guo L., Dong H, & Liu B. (2024). RND1 inhibits epithelial-mesenchymal transition and temozolomide resistance of glioblastoma via AKT/GSK3-β pathway. Cancer Biology & Therapy, 25(1), 2321770.
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9

Immunostaining of MEF Cells with GSNO

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For immunostaining, MEF cells were cultured in 24 wells plate with coverslips. After cell seeding for 24 hours, the cells were further treated with GSNO donor (Sigma Aldrich, 10uM) for 24 hours. Cells were fixed with 4% paraformaldehyde (Sigma Aldrich), and blocked with a blocking solution (normal goat serum) accompanied with 0.1% saponin for permeabilization. After blocking, the cells were incubated in 0.1% saponin with primary antibody: anti-FLAG (Medical & Biological Laboratories), secondary antibody and fluorescence-probe: Alex Flour 633 goat anti-mouse, ProteoStat Dye (1:1000), DAPI (1ug/ml). Images were taken in multi-tracking mode on a laser scanning confocal microscope (LSM710, Carl Zeiss) with a 63 plan apochromat 1.4 NA objective.
Yi W., Zhang Y., Liu B., Zhou Y., Liao D., Qiao X., Gao D., Xie T., Yao Q., Zhang Y., Qiu Y., Huang G., Chen Z., Chen C, & Ju Z. (2021). Protein S-nitrosylation regulates proteostasis and viability of hematopoietic stem cell during regeneration. Cell reports, 34(13), 108922.
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10

Antibody Characterization Protocol

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Anti‐USP11 (catalog no. sc‐365528), anti‐Ub (catalog no. sc‐166553) and anti‐NONO (catalog no. sc‐376865) antibodies were purchased from Santa Cruz. Anti‐USP11 (EPR4346) and anti‐Myc (2276S) antibodies were provided by Abcam. Three antibodies against NONO were obtained from Sangon Biotech (catalog no. D199144), MBL Life science (catalog no. RN092PW), Proteintech Group, Inc (catalog no. 11058‐1‐AP). Anti‐Flag (catalog no. M185‐3L), anti‐HA (catalog no. M180‐3) and anti‐Myc (catalog no.M192‐3) were purchased from Medical & Biological Laboratories. Anti‐GAPDH (catalog no. KC‐5G4) antibody was bought from Kangchen Biotech.
Feng P., Li L., Dai J., Zhou L., Liu J., Zhao J., Li X., Ling N., Qiu S., Zhang L., Xie T., Chen Y., Donovan M.J., Peng T., Song J, & Ye M. (2020). The regulation of NONO by USP11 via deubiquitination is linked to the proliferation of melanoma cells. Journal of Cellular and Molecular Medicine, 25(3), 1507-1517.
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