Anti flag tag antibody

Manufactured by Merck Group

Sourced in United States

The Anti-Flag tag antibody is a laboratory tool used for the detection and purification of proteins tagged with the Flag epitope. It binds specifically to the Flag tag, which is a short polypeptide sequence commonly used as a fusion tag to facilitate the identification and isolation of recombinant proteins. The antibody can be used in various immunoassays and affinity chromatography applications to effectively capture and isolate Flag-tagged proteins from cell lysates or other biological samples.

Automatically generated - may contain errors

Lab products found in correlation

Close spelling variants of this product

Anti-Flag tag mouse monoclonal IgG antibody Anti-FLAG tag antibody-conjugated agarose beads Rabbit anti-Flag tag antibody Anti-Flag-tag rabbit monoclonal antibody Anti-flag tag monoclonal antibody HRP-conjugated mouse anti-FLAG tag antibody HRP-conjugated anti-FLAG tag antibody Mouse monoclonal anti-flag tag antibody Anti-FLAG-tag (M2) antibody Mouse anti-flag tag antibody

17 protocols using anti flag tag antibody

Investigating Anti-Abl Signaling Pathway

Check if the same lab product or an alternative is used in the 5 most similar protocols

SBF‐1 is a synthetic steroidal glycoside, as described previously.¹⁵, ¹⁶ Anti‐c‐Abl, anti‐p‐Bcr, anti‐p‐STAT5, anti‐STAT5, anti‐p‐SHP‐2, anti‐c‐Cbl, anti‐HA‐tag, anti‐myc‐tag, and anti‐Beclin 1 antibodies were purchased from Cell Signaling Technology. Anti‐SHP‐2, anti‐GAPDH, anti‐PTP1B, and anti‐ ubiquitin antibodies were from Santa Cruz Biotechnology. Anti‐p62, anti‐ATG5, and anti‐phosphotyrosine antibodies were from Abcam. MG132 and bafilomycin A1 (Baf A1) were from Selleck Chemicals. Anti‐Flag‐tag antibody, 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT), imatinib, 3‐Methyladenine (3‐MA), chloroquine (CQ), 4′,6‐diamidino‐2‐phenylindole (DAPI), and Oridonin were from Sigma‐Aldrich. The lysosome‐specific dye LysoTraker Red, Lipofectamine™ LTX Reagent, Lipofectamine 2000, and Lipofectamine RNAi MAX were from Life Technologies. SuperSep Phos‐tag™ was from Wako. The RediPlate 96 EnzChek Tyrosine Phosphatase Assay Kit was from Thermo Fisher Scientific.

Elgehama A., Wang Y., Yu Y., Zhou L., Chen Z., Wang L., Sun L., Gao J., Yu B., Shen Y, & Xu Q. (2022). Targeting the PTP1B‐Bcr‐Abl1 interaction for the degradation of T315I mutant Bcr‐Abl1 in chronic myeloid leukemia. Cancer Science, 114(1), 247-258.

+ Open protocol

+ Expand

Characterizing GST-IL-22R Interactions with STAT3

Check if the same lab product or an alternative is used in the 5 most similar protocols

To monitor the interaction between the different GST-IL-22R mutants generated previously^{27 (link)} and STAT3, the proteins were expressed independently in COS-7 cells. Briefly, 4 × 10⁵ COS-7 cells plated in a six-well plate were transiently transfected using ViaFect (Promega), according to the manufacturer’s recommendations. Two days later, cells were lysed in 500 µl of lysis buffer (1% Triton X-100, 10% glycerol, 10 mM Tris (pH 8), 150 mM sodium vanadate, 1 mM sodium fluoride, 5 mM EDTA, 1 mM DTT and cOmplete Protease Inhibitor Cocktail (Sigma)) and cell debris were removed by centrifugation. STAT3 was mixed with recombinant monobody at 10 µM for 8 h at 4 °C. Then, GST-proteins were added for an additional 16 h. Purification of GST was performed using GST SpinTrap columns (GE Healthcare Life Sciences) according to the manufacturer’s recommendations. Input and eluted samples were analyzed by western blot with an anti-STAT3 antibody (12640, CST). The membranes were then reprobed with anti-GST (RPN1236V, Sigma), anti-Flag tag antibody (F1804, Sigma) and anti-His tag antibody (12698, CST).

La Sala G., Michiels C., Kükenshöner T., Brandstoetter T., Maurer B., Koide A., Lau K., Pojer F., Koide S., Sexl V., Dumoutier L, & Hantschel O. (2020). Selective inhibition of STAT3 signaling using monobodies targeting the coiled-coil and N-terminal domains. Nature Communications, 11, 4115.

+ Open protocol

+ Expand

ChIP-seq Analysis Protocol

Cited 2 times

Check if the same lab product or an alternative is used in the 5 most similar protocols

ChIP analysis was conducted as described [33 (link)]. Briefly, DNA was fragmented by sonication to an average size of 400–500 bp for standard ChIP or of 100–200 bp for high-resolution ChIP. Immunoprecipitation was conducted using Dynabeads Protein G (Invitrogen, Carlsbad, CA, USA), anti-FLAG-tag antibody (Sigma-Aldrich, St. Louis, MO, USA; M2), anti-PA-tag antibody (FujiFilm Wako Pure Chemical Corporation, Osaka, Japan; NZ-1), and polyclonal antibody against Rap1 (Santa Cruz Biotechnology, Dallas, TX, USA; yC-19) or Hmo1 [29 (link)]. Real-time quantitative PCR analyses were performed using a KAPA SYBR Fast qPCR kit (Kapa Biosystems, Wilmington, MA, USA). Each experiment was performed in triplicate, and the mean and standard deviation of the ratio of immunoprecipitated DNA to input DNA (IP/input) were calculated. Primer pairs used for quantitative PCR are described in the S1 text.

Kasahara K., Nakayama R., Shiwa Y., Kanesaki Y., Ishige T., Yoshikawa H, & Kokubo T. (2020). Fpr1, a primary target of rapamycin, functions as a transcription factor for ribosomal protein genes cooperatively with Hmo1 in Saccharomyces cerevisiae. PLoS Genetics, 16(6), e1008865.

+ Open protocol

+ Expand

E2 Pellets and Tamoxifen for In Vivo and In Vitro Studies

Check if the same lab product or an alternative is used in the 5 most similar protocols

E2 pellets (0.36-mg 60-day release) for in vivo studies were purchased from Innovative Research (Sarasota, FL). Tamoxifen citrate (Tam) for in vivo studies and 4-hydroxy tamoxifen (Tam) and 17-beta estradiol (E2) for in vitro studies were purchased from Sigma (St Louis, MO). Doxycycline was purchased from Sigma (St Louis, MO). Antibodies against phosphorylated (p)H3, Ki67, and Cleaved Caspase 3–7 were obtained from Millipore (Billerica, MA), Dako (Carpinteria, CA), and Cell Signaling (Danvers, MA) respectively. Antibody against cJun was from Oncogene Research Products (La Jolla, CA). Anti- Flag Tag antibody was from Sigma (St Louis, MO).

Malorni L., Giuliano M., Migliaccio I., Wang T., Creighton C.J., Lupien M., Fu X., Hilsenbeck S.G., Healy N., De Angelis C., Mazumdar A., Trivedi M.V., Massarweh S., Gutierrez C., De Placido S., Jeselsohn R., Brown M., Brown P.H., Osborne C.K, & Schiff R. (2016). Blockade of AP-1 Potentiates Endocrine Therapy and Overcomes Resistance. Molecular cancer research : MCR, 14(5), 470-481.

+ Open protocol

+ Expand

SUMOylation Assay for BCL6 Protein

Check if the same lab product or an alternative is used in the 5 most similar protocols

We used a commercially available SUMOylation Assay Kit (Abcam, San Francisco, CA) according to the manufacturer's instructions. To summarize briefly, THP-1 EV and THP-1-IL-32α were cotransfected with pcDNA3.1 + 5×FLAG-BCL6, and pCS3MT + SUMO-2 and were then treated with 50 nM PMA for 8 h. Cells were lysed in 50 mM HEPES (pH 7.5), 150 mM NaCl, 5% glycerol, 20 mM β-glycerophosphate, 1% NP-40, 0.5% TX-100, and 1 mM EDTA. Extracted whole cells lysates were incubated with anti-flag tag antibody (0.2 μg/well) (Sigma. Louis, MO) for 60 min and then a SUMO detection antibody was added. After incubation, a signal reporter solution was added. Finally, a color developing solution was added and the absorbance was measured at 450 nm by a microplate reader. We calculated SUMOylation of the BCL6 protein as follows.

Park Y.S., Kang J.W., Lee D.H., Kim M.S., Bak Y., Yang Y., Lee H.G., Hong J, & Yoon D.Y. (2014). Interleukin-32α downregulates the activity of the B-cell CLL/lymphoma 6 protein by inhibiting protein kinase Cε-dependent SUMO-2 modification. Oncotarget, 5(18), 8765-8777.

+ Open protocol

+ Expand

Protein Interaction Profiling in HEK293 and THP-1 Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

HEK293, THP-1 EV, and THP-1-IL-32α cells were cotransfected with pcDNA3.1 + 6×Myc-IL-32α, pcDNA3.1 + 5×FLAG-BCL6, and pCS3MT + -SUMO-2 or -ubiquitin and then lysed in 50 mM HEPES (pH 7.5), 150 mM NaCl, 5% glycerol, 20 mM β-glycerophosphate, 1% NP-40, 0.5% TX-100, and 1 mM EDTA. Western blot was performed with an anti-myc tag antibody (Millipore, Bedford, MA), anti-flag tag antibody (Sigma, St. Louis, MO), anti-PKCδ/ε antibody (Santa Cruz Biotechnology, TX), anti-IL-32 antibody KU32-52 [15 (link), 17 (link)], and anti-BCL6 antibody (Santa Cruz Biotechnology). For immunoprecipitation, cell lysates were mixed with 1 μg of anti-myc tag antibody, 1 μg of anti-PKC δ/ε antibody, and 2 μg of anti-flag tag antibody for 1 h and then precipitated with 35 μl of protein G-agarose beads (KPL, Gaithersburg, MD).

+ Open protocol

+ Expand

Overexpression of DNMT3A, DNMT3B, and RING1 in GM12878 Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

Entry vectors bearing DNMT3A, DNMT3B,
or RING1 cDNAs were purchased from the Harvard Plasmid
Repository. DNMT3A or DNMT3B cDNAs were cloned into pLX-TRC313 (Broad Institute)
using Gateway LR Clonase II Enzyme mix (Thermo Fisher Scientific). Control
vector pLX-TRC313-GFP was obtained from Broad Institute. GM12878 LCLs were
transduced with pLX-TRC313-GFP, -DNMT3A, or -DNMT3B encoding lentiviruses. Cells
were selected with hygromycin for 2 weeks. Heterogenous protein expression was
confirmed by immunoblot using anti-V5 tag antibody (V8012, Sigma-Aldrich).
RING1 cDNA was cloned into pHAGE-3×FLAG-MYC vector
using Gateway LR Clonase II Enzyme mix (Thermo Fisher Scientific). Control
vector pHAGE-GFP was obtained from Addgene (plasmid #106281). GM12878 LCLs were
transduced with either pHAGE-GFP or RING1 encoding lentiviruses. Cells were
selected with puromycin for 4 days. Heterogenous protein expression was
confirmed by immunoblot using anti-Flag tag antibody (F1804, Sigma-Aldrich).

Guo R., Zhang Y., Teng M., Jiang C., Schineller M., Zhao B., Doench J.G., O’Reilly R.J., Cesarman E., Guilino-Roth L, & Gewurz B.E. (2020). DNA Methylation Enzymes and PRC1 Restrict B-cell Epstein-Barr Virus Oncoprotein Expression. Nature microbiology, 5(8), 1051-1063.

+ Open protocol

+ Expand

Membrane Protein Detection in Bacterial Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

Cells (5 mg of wet weight) were harvested after 16 h-20 h growth on salt-free LB agar with 0.1 % arabinose and appropriate antibiotics at 28 °C. For detection of the membrane proteins BcsA and BcsC, 200 μl of urea buffer (8 M urea, 2% SDS, 11% glycerol, 62.5 mM Tris-HCl, pH 6.8) was added and sonicated four times (10 s, 3.5 μm amplitude) on ice as described previously [76 (link); 77 (link)]. The sonicated extracts were examined with SDS-PAGE gels stained with Coomassie Blue to ensure loading of equal amounts of all sample proteins for the Western blots (with the exception of samples in Fig. 2B, where 10 times higher amounts of the cell extract from the bcsG mutant had to be loaded owing to the approx. 10-fold lower BcsA expression in the mutant cells). For Western blots, cell extracts were separated on SDS-PAGE gel and transferred onto a PVDF membrane (Millipore). BcsA-3xFLAG, BcsC-3xFLAG, and BcsG-3xFLAG were detected by using anti-FLAG-tag antibody (1:2000 for BcsA, 1:1000 for BcsC, and 1:2000 for BcsG; Sigma) and peroxidase-conjugated AffiniPure Goat Anti-Mouse IgG (1:3000, Jackson ImmuniResearch). Visualization of bands was performed using FUJI LAS1000-plus chemiluminescence imaging system (Fuji, Stamford, CT, USA).

Sun L., Vella P., Schnell R., Polyakova A., Bourenkov G., Li F., Cimdins A., Schneider T.R., Lindqvist Y., Galperin M.Y., Schneider G, & Römling U. (2018). Structural and functional characterization of the BcsG subunit of the cellulose synthase in Salmonella typhimurium. Journal of molecular biology, 430(18 Pt B), 3170-3189.

+ Open protocol

+ Expand

ADAMTS1 and APP Interaction Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols

HEK 293T cells co-transfected with ADAMTS1-FLAG and APP-Myc were washed with PBS buffer and solubilized in ice-bath by CelLytic lysis buffer (C2978, Sigma-Aldrich, Shanghai, China) containing protease inhibitors. Clear lysates were incubated with primary antibodies overnight at 4°C and then with protein G Agarose beads (11243233001, Roche, Basel, Switzerland) for 2 h at 4°C. After five-time rinse with TBS buffer, immunoprecipitated proteins were recovered from the beads by boiling for 5 min in sample buffer, and then analyzed by immunoblotting.
Antibodies were diluted with TBST buffer containing 5% skim milk and listed below: anti-FLAG tag antibody (1:5,000, RRID:AB_262044, F1804, Sigma-Aldrich, Burlington, United States), anti-Myc tag antibody (1:5,000, RRID:AB_331783, 2276, Cell Signaling Technology, Danvers, United States) and anti-Na⁺/K⁺ ATPase antibody (1:5,000, RRID:AB_2227873, 14418-1-AP, Proteintech, Wuhan, China).

Qiu Y., Sha L., Zhang X., Li G., Zhu W, & Xu Q. (2022). Induction of A Disintegrin and Metalloproteinase with Thrombospondin motifs 1 by a rare variant or cognitive activities reduces hippocampal amyloid-β and consequent Alzheimer’s disease risk. Frontiers in Aging Neuroscience, 14, 896522.

+ Open protocol

+ Expand

Coimmunoprecipitation and Competition Experiments in COS-7 Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

Coimmunoprecipitation assays in COS-7 cells were performed with agarose beads conjugated with anti-FLAG tag antibody (Sigma-Aldrich) as described previously44 (link)52 (link). Immunoprecipitated proteins and coimmunoprecipitated proteins were analyzed by 10% SDS-PAGE followed by immunoblotting with specific antibodies.
Competition experiments between Rab1A and Arl8b were performed with glutathione Sepharose 4B beads coupled with T7-GST-SKIP-RUN (GE Healthcare Ltd., Little Chalfont, UK) essentially as described previously53 (link). In brief, T7-GST-SKIP-RUN was transiently expressed in COS-7 cells and affinity-purified with glutathione Sepharose beads. Beads coupled with T7-GST-SKIP-RUN or beads alone were incubated for 3 hours at 4°C with COS-7 cell lysates containing FLAG-Rab1A(Q70L) and/or FLAG-Arl8b(Q75L). The proteins bound to the beads were analyzed by 11.25% SDS-PAGE followed by immunoblotting with specific antibodies. The intensity of the immunoreactive bands in Fig. 5A was measured with ImageJ software.

Ishida M., Ohbayashi N, & Fukuda M. (2015). Rab1A regulates anterograde melanosome transport by recruiting kinesin-1 to melanosomes through interaction with SKIP. Scientific Reports, 5, 8238.

+ Open protocol

+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!