Flag tag (flag)

Manufactured by Sangon

Sourced in China

FLAG is a laboratory equipment designed for the purification and detection of recombinant proteins. It is used to facilitate the isolation and identification of target proteins that have been engineered to include a specific FLAG epitope tag.

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Lab products found in correlation

6 protocols using flag tag (flag)

Antibody-based Mitochondrial Protein Analysis

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The antibodies used in our experiments included: ACAT1 (CST, 44276), acetylated-lysine (CST, 9441), BAX (Proteintech, 50599-2-Ig), BCL-2 (Proteintech, 12789-1-AP), COXⅣ (CST, 11967), cleaved-PARP-1 (CST, 5625), cleaved-caspase3 (CST, 9661), EP300 (Abcam, ab14984), FLAG (Sangon Biotech, D191041), anti-FLAG® M2 Affinity Gel (MERCK, F2426), GAPDH (Proteintech, 600004-1-lg), HDAC1 (Proteintech, 10197-1-AP), HDAC2 (Proteintech, 12922-3-AP), HDAC3 (Proteintech, 10255-1-AP), HSP60 (CST, 12165), HA (Sangon Biotech, D110004), Ki67 (Proteintech, 27309-1-AP), LC3 (MERCK, L7543), MFN2 (Proteintech, 12186-1-AP), PINK1 (CST, 6946), Parkin (Proteintech, 14060-1-AP; CST, 4211), Parkin (phospho-Ser65, Biorbyt, orb312554), P62 (MERCK, P0067), TOMM20 (CST, 42406), TIM23 (Santa Cruz Biotechnology, sc-514463), ubiquitin (Proteintech, 10201-2-AP), ULK1 (CST, 8054), VDAC1 (Proteintech, 10866-1-AP), α-tubulin (Sigma, T6199), β-actin (ABclonal, AC004).
The chemicals used in our experiments were: bafilomycin A1 (Sigma, B1793), chloroquine (MedChemExpress, HY-17589A), Hoechst (Beyotime, 33342), suberoylanilide hydroxamic acid (SAHA; MedChemExpress, HY-10221), and trichostatin A (TSA, MedChemExpress, HY-15144).

Sun X., Shu Y., Ye G., Wu C., Xu M., Gao R., Huang D, & Zhang J. (2021). Histone deacetylase inhibitors inhibit cervical cancer growth through Parkin acetylation-mediated mitophagy. Acta Pharmaceutica Sinica. B, 12(2), 838-852.

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Macrophage Protein Profiling by Western Blot

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Protein lysates were harvested from macrophages using lysis buffer containing 50 mM Tris–Cl pH 7.4, 150 mM NaCl, 1% NP-40, 1 mM EDTA-free protease inhibitor cocktail (Roche) and 1 mM phenylmethylsulfonyl fluoride (PMSF). 50 μg of lysates were separated by SDS-PAGE and transferred to PVDF membrane (Millipore) for western blot analysis with specific primary antibodies (1:1000) for Ubiquitin (abcam, cat#ab13493), Msl2(CST,cat#ab44006), Outlin (abcam, cat# ab211328), p62 (Cell Signaling Technology, cat#ab16177), PI3Kp110α (Cell Signaling Technology,cat#ab4255), PI3K-p110β(Cell Signaling Technology,cat#ab3011), PI3Kp110δ(Cell Signaling Technology,cat#ab5405), Vps34(Cell Signaling Technology,cat#ab4263, AKT(Cell Signaling Technology,cat#ab4685), p-AKT(Ser473) (Cell Signaling Technology,cat#ab4060), mTOR(Cell Signaling Technology,cat#ab2972), Phospho-mTOR (Ser2448) (Cell Signaling Technology,cat#ab5563) and LC3 (Cell Signaling Technology, cat#ab4108), QKI (Sigma), LC3-II(Cell Signaling Technology, cat#ab2775), flag(Sangon, cat#D191041), HA(Sangon, cat#D191044), and Myc(Sangon, cat#D199941), Rnf6 (Thermo, cat#PA5-59044), EDC4 (proteintech, cat#D17737-1-AP) and β-actin(Sangon, cat#D191047). Immunolabelled proteins were detected by using appropriate HRP-conjugated secondary antibodies (Thermo, cat#31460), followed by visualization with ECL (Sangon).

Zhai D., Wang W., Ye Z., Xue K., Chen G., Hu S., Yan Z., Guo Y., Wang F., Li X., Xiang A., Li X., Lu Z, & Wang L. (2022). QKI degradation in macrophage by RNF6 protects mice from MRSA infection via enhancing PI3K p110β dependent autophagy. Cell & Bioscience, 12, 154.

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Phosphorylation-Dependent Regulation of PPARδ

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PPARδ (Cat: SC-74440) was obtained from Santa Cruz. GAPDH (Cat: 60004) was obtained from Proteintech. p62 (Cat: ab56416) was obtained from Abcam. Actin (Cat: D195316), GST (Cat: D110271), and Flag (Cat: D110005) were purchased from Sangon Biotech. AMPKα (RLT026), phospho-AMPKα1/2 (RLP0575), and anti-phosphoserine (RLM3440) were purchased from Ruiying Biological. The p-PPARδ-S50 mouse polyclonal antibody was developed by using human PPARδ peptide PSSSYTDLSRSSSpPPSLLDQL (nonphosphorylation peptide as a negative control), which was purchased from Chinese Peptide Company. Western blot analysis showed that the p-PPARδ-S50 antibody did not cross-react with other PPAR family members (Fig. S1). Secondary antibodies were obtained from Jackson ImmunoResearch. Cells were lysed in the lysis buffer (50 mM Tris HCl, pH 7.4, 250 mM NaCl, 0.5% Triton X-100, PMSF, 10% glycerol, protease inhibitor cocktail). Protein concentration was determined by using the Pierce BCA Protein Assay Kit (Thermo). The samples were subjected to SDS-PAGE, transferred to a nitrocellulose membrane, and then probed by the indicated antibodies and developed by using an ECL reagent. Blots were quantified by using ImageJ.

Ding J., Gou Q., Jia X., Liu Q., Jin J., Shi J, & Hou Y. (2021). AMPK phosphorylates PPARδ to mediate its stabilization, inhibit glucose and glutamine uptake and colon tumor growth. The Journal of Biological Chemistry, 297(3), 100954.

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NanoSwitches Peptide Binding Assay

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For the competition assay of the NanoSwitches, peptides of Flag tag (DYKDDDDK), HA tag (YPYDVPDYA), PG4 (LQPELDSFKEELDKYFKNH TSPDVD), and P21 (PSKPSKRSFIEDLLFNKV) were synthesized (Sangon, Shanghai, China). Purified recombinant proteins were incubated with COVID-19 serum or antibodies in reaction systems containing 50 mM HEPES (pH 7.5), 3 mM EDTA, 150 mM NaCl, 0.005% (vol/vol) Tween-20, 10 mM DTT and different concentrations of synthetic peptides. The systems were conducted at 37 °C for 60 min, and luminescence was detected using a Nano-Glo® Luciferase Assay System (Promega, USA) following the manufacturer's instructions.

Li J., Wang J.L., Zhang W.L., Tu Z., Cai X.F., Wang Y.W., Gan C.Y., Deng H.J., Cui J., Shu Z.C., Long Q.X., Chen J., Tang N., Hu X., Huang A.L, & Hu J.L. (2022). Protein sensors combining both on-and-off model for antibody homogeneous assay. Biosensors & Bioelectronics, 209, 114226.

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SARS-CoV-2 Propagation and Cell Line Maintenance

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Human colorectal adenocarcinoma epithelial cell Caco-2, human kidney epithelial cell derived HEK293T-hACE2, and African green monkey kidney epithelial cell Vero E6 were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 1% penicillin-streptomycin and 10% FBS (Gibco). The HEK293T-hACE2 cell was kindly provided by Zhong Ji Dang Kang Biotechnology Co., Ltd., Beijing, China. All cell lines were cultured at 37 °C with 5% CO₂. SARS-CoV-2 isolate CHN/Beijing_IME-BJ01/2020 (GenBank accession no. MT291831.1), originated from human throat swabs, was propagated in Vero E6 cells in DMEM (HyClone) supplemented with 2% FBS, and titred using a tissue culture median infectious dose assay (TCID₅₀). All experiments of infectious SARS-CoV-2 were conducted under biosafety level 3 facilities. HA-tagged SIRT5 plasmid was purchased from Miaoling Biotech, and the SARS-CoV NSP14 was cloned into vector VR1012 with the Flag-tag (Sangon Biotech) and confirmed by sequencing.

Liu Q., Wang H., Zhang H., Sui L., Li L., Xu W., Du S., Hao P., Jiang Y., Chen J., Qu X., Tian M., Zhao Y., Guo X., Wang X., Song W., Song G., Wei Z., Hou Z., Wang G., Sun M., Li X., Lu H., Zhuang X., Jin N., Zhao Y., Li C, & Liao M. (2022). The global succinylation of SARS-CoV-2–infected host cells reveals drug targets. Proceedings of the National Academy of Sciences of the United States of America, 119(30), e2123065119.

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Characterization of SARS-CoV-2 M Protein Interactions

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The SARS-CoV M protein (NC_004718), SARS-CoV-2 M protein of IPBCAMS-WH-01/2019 strain (no. EPI_ISL_402123), TBK1 genes and their truncations were cloned into vector VR1012 with the Flag-tag or GST-tag (Sangon Biotech, Shanghai, China). The expression vectors of Flag-Ubi, Flag-K48-Ubi, Flag-K63-Ubi, pIFNβ-Luc, ISRE-luc and Renilla luc were constructed in the previous study (17 (link), 18 (link)). The expression plasmids for IRF3, TANK, IKKϵ, RIG-I and TRAF3 were purchased from PPL Biotech, Jiangsu, China. The expression plasmid for TBK1 and MDA5 was purchased from Miaoling Biotech, Wuhan, China.

Sui L., Zhao Y., Wang W., Wu P., Wang Z., Yu Y., Hou Z., Tan G, & Liu Q. (2021). SARS-CoV-2 Membrane Protein Inhibits Type I Interferon Production Through Ubiquitin-Mediated Degradation of TBK1. Frontiers in Immunology, 12, 662989.

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