Flag tag (flag)

Manufactured by OriGene

Sourced in United States

The FLAG is a laboratory equipment product designed to facilitate protein purification and analysis. It serves as an affinity tag that can be attached to recombinant proteins, allowing for their selective capture and isolation from complex samples. The FLAG tag is a short peptide sequence that can be recognized by specific antibodies, enabling efficient detection and purification of the tagged proteins.

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Lab products found in correlation

Pvdf membrane, by Merck Group (2 mentions) Acetylated lysine, by Cell Signaling Technology (1 mentions) Sirt6, by Cell Signaling Technology (1 mentions) Digoxigenin, by Merck Group (1 mentions) Digoxin, by Merck Group (1 mentions) Rapamycin, by Merck Group (1 mentions) Lamp1, by Abcam (1 mentions) Protein a magnetic beads, by Thermo Fisher Scientific (1 mentions) Dapi mounting media, by Merck Group (1 mentions) Keap1, by OriGene (1 mentions) Caspase 3, by Santa Cruz Biotechnology (1 mentions) Cleaved caspase 3, by Cell Signaling Technology (1 mentions) N acetylcysteine, by Merck Group (1 mentions) Actinomycin d, by Merck Group (1 mentions) Ubiquitin, by Merck Group (1 mentions) β actin, by Abcam (1 mentions) Survivin, by R&D Systems (1 mentions) Nitrocellulose membrane, by Macherey-Nagel (1 mentions) Cyclin d1, by Cell Signaling Technology (1 mentions) Ez ecl kit, by Sartorius (1 mentions)

10 protocols using flag tag (flag)

BDV-N and NF-κB Protein Interaction

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For the IP assay, 293T cells were transfected with pcDNA3 encoding BDV-N, alanine substitution mutants in the first 8 (F8A) or last 8 (L8A) amino acids of IPBN, or IPBN deletion mutants of BDV-N tagged with HA at N-terminus, pCMV6 expressing NF-κB1 fused with FLAG-tag at C-terminus (OriGene Technologies, Inc., Rockville, MD, USA), and pCMV6 expressing NF-κB2 fused with FLAG-tag at C-terminus (OriGene Technologies, Inc.), and the cells were then lysed in a buffer containing 20 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% Triton-X, 1 mM EDTA, and 1 X complete protease inhibitor cocktail. Cell lysate expressing HA-tagged BDV-N or BDV-N mutants was mixed with cell lysate transfected with FLAG-tagged NF-κB1 or FLAG-tagged NF-κB2 for 30 min at 4°C, and the mixed cell lysates were immunoprecipitated with anti-FLAG M2 monoclonal antibody using Protein G Dynabeads, according to the manufacturer's instructions. The mixed cell lysates and IP products were subjected to SDS-PAGE and western blotting using an anti-HA monoclonal antibody (Abcam, Cambridge, UK) and an anti-FLAG M2 monoclonal antibody.

Makino A., Fujino K., Parrish N.F., Honda T, & Tomonaga K. (2015). Borna disease virus possesses an NF-ĸB inhibitory sequence in the nucleoprotein gene. Scientific Reports, 5, 8696.

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Immunoprecipitation Assay Protocol

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IP assay was conducted following the IP protocol utilizing magnetic separation which provided by Cell Signaling using antibodies against flag (1:200 OriGene), SIRT6 (1:200 Cell Signaling), or acetylated-lysine (1:100 Cell Signaling). The samples were boiled in 5 X SDS-PAGE sample buffer and subjected to immunoblot analysis

Sugatani T., Agapova O., Malluche H.H, & Hruska K.A. (2015). SIRT6 Deficiency Culminates in Low-Turnover Osteopenia. Bone, 81, 168-177.

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Autophagy and Apoptosis Regulation

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Chloroquine, ammonium chloride, digoxin, digoxigenin, strophanthidin, DAPI mounting media, rapamycin, 3‐(4,5‐dimethylthiazol‐2yr)‐2‐5‐diphenyltetrazoliumbromide (MTT), actinomycin D, bafilomycin SMER28 and N‐acetylcysteine from Sigma (Poole, UK) were used.
Protein A magnetic beads were purchased from Thermo Scientific (East Riding of Yorkshire, UK). Antibodies against the following were used for immunoblotting: p62 (rabbit polyclonal; #P0067), LC3 (rabbit polyclonal; #L8918), Ubiquitin (rabbit polyclonal; #SAB1306222), NBR1 (rabbit polyclonal, #SAB2107031) from Sigma. p62 (mouse monoclonal; # sc‐28359), caspase‐3 (rabbit polyclonal; #sc‐136219) from Santa Cruz Biotechnology. Beta‐actin (mouse monoclonal; #ab8226), GFP (#ab1218), and LAMP1 (rabbit polyclonal; #ab24170) from Abcam. Cleaved caspase‐3 (rabbit polyclonal; #9661) from Cell Signalling (New England Biolabs). Keap1 (mouse monoclonal; #TA502059) and FLAG (mouse monoclonal; #TA50011‐100) from Origene).

Mercado N., Colley T., Baker J.R., Vuppussetty C., Kono Y., Clarke C., Tooze S., Johansen T, & Barnes P.J. (2019). Bicaudal D1 impairs autophagosome maturation in chronic obstructive pulmonary disease. FASEB bioAdvances, 1(11), 688-705.

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Western Blot Analysis of Cell Signaling

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Cells were harvested, lysed with RIPA and 30 μg of total protein were separated on a 10% SDS-PAGE gel. Gels were transferred to nitrocellulose membranes (Macherey-Nagel) and probed with primary antibodies targeting Flag (mouse monoclonal; 1:4000; OriGene), β-actin (goat polyclonal; 1:2000; Santa Cruz Biotechnology), P-FAK (rabbit monoclonal; 1:1000; Cell Signaling), FAK (mouse monoclonal; 1:1000; Santa Cruz Biotechnology), cyclin D1 (rabbit monoclonal; 1:1000; Cell Signaling) and survivin (rabbit polyclonal; 1:2000; R&D Systems). After incubation with an HRP-conjugated mouse anti-IgG (Santa Cruz, 1:2000), membranes were revealed using EZ-ECL kit (Biological Industries, Haemek, Israel) and analyzed on a ChemiDoc Imaging System (Bio-Rad).

Pérez-Moreno P., Quezada-Meza C., Chavez-Almarza C., Niechi I., Silva-Pavez E., Trigo-Hidalgo C., Aguayo F., Jara L., Cáceres-Verschae A., Varas-Godoy M., Díaz V.M., García de Herreros A., Burzio V.A, & Tapia J.C. (2020). Phosphorylation of Endothelin-Converting Enzyme-1c at Serines 18 and 20 by CK2 Promotes Aggressiveness Traits in Colorectal Cancer Cells. Frontiers in Oncology, 10, 1004.

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Western Blot Analysis of Cell Lysates

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Cell lysates were prepared in RIPA buffer with protease inhibitors and then separated on 12% SDS-PAGE gels. The separated proteins were electroblotted onto PVDF membranes (Millipore, USA). The membrane was blocked with 5% milk in TBST and then incubated with specific primary antibodies: LaminA/C (1:1000, ab169532, Abcam, USA), Paxillin (1:1000, #2542, Cell Signaling, USA), Flag (1:1000, #TA180144, OriGene, USA), N-cadherin (1:2000, 66219-1-Ig, Proteintech, USA), E-cadherin (1:5000, 20874-1-AP, Proteintech, China), vimentin (1:10000, 60330-1-1g, Proteintech, USA), and GAPDH (1:10000, 60004-1-Ig, Proteintech, USA). The primary antibody incubation was performed at 4°C. Subsequently, the membrane was incubated with either HRP-conjugated secondary antibody mouse (1:2000, A0216, Beyotime, China) or secondary antibody rabbit (1:2000, A0208, Beyotime, China) at room temperature. Finally, the protein bands were visualized using the ECL Western Blot Analysis System (Pierce, USA).

Liu W., Huang X., Luo W., Liu X, & Chen W. (2024). Progerin Inhibits the Proliferation and Migration of Melanoma Cells by Regulating the Expression of Paxillin. OncoTargets and Therapy, 17, 227-242.

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Overexpression and ChIP analysis of EGR1

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Human EGR1 was amplified and cloned into the pCMV6-ACIRES-GFP-Puro vector to tag EGR1 with Myc and FLAG (Origene Technologies, Rockville, MD, USA). 293 T cells were transfected with an EGR1-MYC-FLAG expression plasmid for 48 h, washed once with PBS, and added to 10 ml of PBS containing 1% formaldehyde to covalently cross-link any DNA–protein complexes at room temperature for 10 min. The beads were added to the samples, rotated for 30 min, and collected by centrifugation at 12,000×g for 1 min. The elution buffers were added to the samples and the supernatants were transferred to clean microcentrifuge tubes. The DNA samples were used for PCR reactions with the appropriate primers.

Park M., Park S.H., Park H., Kim H.R., Lim H.J, & Song H. (2021). ADAMTS-1: a novel target gene of an estrogen-induced transcription factor, EGR1, critical for embryo implantation in the mouse uterus. Cell & Bioscience, 11, 155.

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Immunoblotting Analysis of IDH Mutations

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The tissue specimens or cell pellets were lysed with Radioimmunoprecipitation assay buffer (RIPA buffer, Thermo Fisher) and quantified using the DC Protein Assay (Bio-Rad). Protein samples were resolved on 4-12% Bis-tris gel (Thermo Fisher) and transferred to PVDF membrane (MilliPore). The membranes were labeled with primary antibodies and visualized through chemiluminescence kit (Bio-Rad). The primary antibodies used in this study include anti-IDH1/2 Mutant (R132/R172), clone MsMab-1 (1:2,000, MilliPore); Flag (1:5,000, Origene); and β-actin (1:5,000, Cell Signaling Technology).

Lang F., Jha A., Meuter L., Pacak K, & Yang C. (2021). Identification of Isocitrate Dehydrogenase 2 (IDH2) Mutation in Carotid Body Paraganglioma. Frontiers in Endocrinology, 12, 731096.

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Purification of Human NEU1 and NEU4

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Plasmids encoding FLAG-tagged human NEU1 and NEU4 were purchased from OriGene. DNA were transfected into HEK293F cells in suspension culture using a 1:3 ratio of DNA to polyethyleneimine (1 mg/ml). An equal volume of fresh culture media supplemented with 2.2 mM valproic acid was added 16 to 18 h after transfection, and the cells were allowed to grow for 2 to 3 additional days and then harvested. Cells were lysed with 1x tris buffered saline (TBS), pH 7.4, supplemented with 1x EDTA-free Halt protease inhibitors (Thermo) and 0.1% Triton X-100. FLAG-tagged NEU1 and NEU4 were purified from whole-cell lysates using FLAG agarose (Sigma). Eluates obtained from using 0.15 mg/ml 3 x FLAG peptide (AnaSpec) dissolved in 1x TBS, pH 7.4, were concentrated using a 10-kDa MWCO spin concentrator and diafiltrated to remove FLAG peptide. Concentrated proteins (NEU1-1.99 mg/ml, NEU4-2.3 mg/ml) were quantified using the Bradford assay and stored at 4 °C and used within a month or flash-frozen and stored at −80 °C for extended durations.

Selvan N., Mehta N., Venkateswaran S., Brignol N., Graziano M., Sheikh M.O., McAnany Y., Hung F., Madrid M., Krampetz R., Siano N., Mehta A., Brudvig J., Gotschall R., Weimer J.M, & Do H.V. (2021). Endolysosomal N-glycan processing is critical to attain the most active form of the enzyme acid alpha-glucosidase. The Journal of Biological Chemistry, 296, 100769.

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Caspase-3 Resistant GSAP Mutant

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A putative consensus cleavage site specific for caspase3 was identified in the GSAP-FL amino acid sequence and located into the GSAP-16kDa C-terminal fragment (737DLD739). To obtain a caspase3 resistant GSAP mutant, the required aspartic acids (D) in position 737 and 739 were substituted with alanine (A) and glutamic acid (E), respectively, by direct mutagenesis (Origene, Rockville, MD). In addition, both wild type and mutated GSAP were DDK tagged (FLAG tag) (Origene, Rockville, MD).

Chu J., Li J.G., Joshi Y.B., Giannopoulos P.F., Hoffman N.E., Madesh M, & Praticò D. (2014). Gamma secretase activating protein is a substrate for caspase-3: implications for Alzheimer’s disease. Biological psychiatry, 77(8), 720-728.

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Protein Immunoprecipitation and Western Blot Analysis

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Immunoprecipitation was performed per manufacturer's instructions. Cell pellets were lysed in RIPA buffer with Protease Inhibitor Cocktail and 0.5% SDS. Total cell lysate was precipitated using the DynaBeads Protein G Immunoprecipitation Kit (Invitrogen) and antibodies against FLAG-tag (Origene, Rockville, Maryland, USA) or HA (Sigma-Aldrich, St. Louis, Missouri, USA). Precipitated protein was eluted and resolved by Western blot analysis. Three independent experiments were performed for each representative immunoblot provided in the figures. Normalized densitometric values (protein/loading control) were obtained and displayed.

Zhang C., Yang C., Feldman M.J., Wang H., Pang Y., Maggio D.M., Zhu D., Nesvick C.L., Dmitriev P., Bullova P., Chittiboina P., Brady R.O., Pacak K, & Zhuang Z. (2017). Vorinostat suppresses hypoxia signaling by modulating nuclear translocation of hypoxia inducible factor 1 alpha. Oncotarget, 8(34), 56110-56125.

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