Anti flag antibody clone m2

Manufactured by Merck Group

Sourced in Germany

The Anti-FLAG antibody (clone M2) is a laboratory reagent used to detect and purify proteins that have been engineered to contain a specific amino acid sequence known as the FLAG tag. The antibody binds to this tag, allowing for the identification and isolation of the tagged protein from complex mixtures. This antibody is a commonly used tool in molecular biology and protein research.

Automatically generated - may contain errors

Lab products found in correlation

Nupage sample buffer, by Thermo Fisher Scientific (2 mentions) Immobilon fl pvdf membrane, by Merck Group (2 mentions) Irdye 800cw, by LI COR (2 mentions) Sds page gel, by Thermo Fisher Scientific (2 mentions) Irdye 680rd, by LI COR (2 mentions) Nupage sample reducing agent, by Thermo Fisher Scientific (2 mentions) Mes buffer, by Thermo Fisher Scientific (2 mentions) Tev protease cleavage, by Thermo Fisher Scientific (2 mentions) Protein g magnetic beads, by Thermo Fisher Scientific (2 mentions) Fastprep 24, by MP Biomedicals (2 mentions) Zeba desalting column, by Thermo Fisher Scientific (2 mentions) Streptavidin agarose cl 6b, by Thermo Fisher Scientific (2 mentions) Quizartinib, by MedChemExpress (1 mentions) Sorafenib, by Selleck Chemicals (1 mentions) Anti pim1, by Cell Signaling Technology (1 mentions) Anti bad, by Cell Signaling Technology (1 mentions) Anti flag m2 affinity gel, by Merck Group (1 mentions) Tunicamycin, by Merck Group (1 mentions) Brefeldin a bfa, by Merck Group (1 mentions) U0126, by Merck Group (1 mentions)

28 protocols using anti flag antibody clone m2

FLT3 Inhibitor Screening Protocol

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

The polyclonal rabbit anti-FLT3 (C-20) antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-phospho-FLT3 (Tyr591), anti-phospho-STAT5 (Tyr694), anti-phospho-AKT (Ser473), anti-phospho-p44/42 MAPK (Thr202/Tyr204), anti-phospho-BAD (Ser112), anti-AKT, anti-MAPK, anti-BAD, anti-PIM1 and Sepharose Conjugated Myc-tag antibodies were purchased from Cell Signaling Technology (Beverly, MA). Anti-STAT5 antibody was from BD Transduction Laboratories (San Jose, CA). Anti-FLAG antibody (clone M2) and anti-FLAG M2 affinity gel were from Sigma-Aldrich (St.Louis, MO). Anti-mouse and anti-rabbit horseradish peroxidase antibodies were purchased from GE Healthcare (Buckinghamshire, UK).
Five FLT3 inhibitors were used in this study: quizartinib was purchased from MedChemExpress (Mommouth Junction, NJ), sorafenib was from Selleck Chemicals (Houston, TX), KW-2449 [33 (link)] was a generous gift from Kyowa Hakko Kirin Co., Ltd. (Tokyo, Japan), midostaurin (PKC412) was from Enzo Life Sciences (Plymouth Meeting, PA), lestaurtinib (CEP701) and U0126 were from Merck Millipore Corporation (Billerica, MA). Tunicamycin and brefeldin A (BFA) were purchased from Sigma-Aldrich. Recombinant human FLT3 ligand (FL) was purchased from R&D Systems, Inc.

Chen F., Ishikawa Y., Akashi A., Naoe T, & Kiyoi H. (2016). Co-expression of wild-type FLT3 attenuates the inhibitory effect of FLT3 inhibitor on FLT3 mutated leukemia cells. Oncotarget, 7(30), 47018-47032.

+ Open protocol

+ Expand

Stefin B Expressing iMACs Immunoblot

Cited 1 time

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

Samples for analysis of the Stefin B expressing iMACs were prepared and the immunoblot performed as described previously ^{49 (link)}. Briefly cells were harvested, washed once in PBS and then lysed in RIPA lysis buffer (cOmplete protease inhibitors (Roche)), then sonicated to disrupt DNA to facilitate gel loading. The samples were then denatured and reduced by adding NuPAGE sample buffer (25% sample volume; Life technologies) and NuPAGE sample reducing agent (10% sample volume; Life technologies) and heating the samples to 95°C for 5 min. Proteins were separated by electrophoresis using a precast 4-12% SDS-PAGE gel (Novex, Invitrogen) and MES buffer (Novex, Invitrogen). The proteins were transferred to an Immobilon-FL PVDF membrane (Millipore) and blocked using 3% BSA in Tris-buffered saline pH7.4 (BSA-TBS) for 1h. The membrane was then probed with an anti-Flag antibody (Clone M2, Sigma) at a 1:1000 dilution and an anti-tubulin antibody (CST) at a 1:1000 dilution in BSA-TBS with 0.1% Tween overnight at 4C. Secondary antibodies (coupled to IRDye 680RD ot IRDye 800CW, Li-Cor Biosciences) were used 1:25,000 dilution.

Selkrig J., Li N., Hausmann A., Mangan M.S., Zietek M., Mateus A., Bobonis J., Sueki A., Imamura H., El Debs B., Sigismondo G., Florea B.I., Overkleeft H.S., Kopitar-Jerala N., Turk B., Beltrao P., Savitski M., Latz E., Hardt W.D., Krijgsveld J, & Typas A. (2020). Spatiotemporal proteomics uncovers cathepsin-dependent macrophage cell death during Salmonella infection. Nature microbiology, 5(9), 1119-1133.

+ Open protocol

+ Expand

Detecting Protein Expression by Western Blot

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

For each sample, three leaf discs were harvested 48 hr after agro‐infiltration to detect protein expression levels by western blot. Samples were ground in 100 μl NuPAGE LDS sample buffer with NuPAGE Reducing Agent as per the supplier's instructions (Life Technologies) and centrifuged at 15,700 × g for 10 min, and the supernatant was collected. After heating at 70 °C for 10 min, total protein extracts were separated on NuPAGE 4%–12% Bis‐Tris Mini Protein Gels (Thermo Fisher) and transferred to nitrocellulose membranes (Millipore) for immunoblotting. HA‐tagged proteins were detected using a horseradish peroxidase (HRP)‐conjugated anti‐HA antibody (clone 3F10; Roche), while the Pikp‐1:Flag protein was detected using an anti‐FLAG antibody (clone M2; Sigma‐Aldrich) and a secondary HRP‐conjugated anti‐mouse antibody (Sigma‐Aldrich). The Immobilon Western kit (Millipore) was used for detection.

Xi Y., Chochois V., Kroj T, & Cesari S. (2021). A novel robust and high‐throughput method to measure cell death in Nicotiana benthamiana leaves by fluorescence imaging. Molecular Plant Pathology, 22(12), 1688-1696.

+ Open protocol

+ Expand

Reagents for Influenza and Glycan Studies

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

NA from C. perfringens (type V), KDO, 4-MUNANA, complete Freund’s adjuvant (CFA), incomplete Freund’s adjuvant (IFA), and anti-FLAG antibody (clone M2) were purchased from Sigma-Aldrich (St. Louis, MO). Anti-HA tag antibody was purchased from Roche Applied Science (Indianapolis, IN). Anti-NEU1 antibody was purchased from Rockland (Gilbertsville, PA). Zanamivir was purchased from GlaxoSmithKline (Research Triangle Park, NC). Oseltamivir phosphate (Tamiflu) was purchased from Sequoia Research Products (Berkshire, United Kingdom). 2-DN was purchased from Calbiochem (Gibbstown, NJ). Goat antiserum against influenza virus serotypes N1 to N9 was obtained from BEI Resources (Manassas, VA).

Feng C., Li J., Snyder G., Huang W., Goldblum S.E., Chen W.H., Wang L.X., McClane B.A, & Cross A.S. (2017). Antibody against Microbial Neuraminidases Recognizes Human Sialidase 3 (NEU3): the Neuraminidase/Sialidase Superfamily Revisited. mBio, 8(3), e00078-17.

+ Open protocol

+ Expand

Affinity Purification of Chromatin Remodelers

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

RH4 cells expressing endogenous 3xFlag tagged CHD4 (both N- and C-terminus) or 3xFlag tagged BRD4 (N-terminus) were grown to confluency in 15 cm dishes. Per condition, three confluent dishes were used. Cells were washed with PBS, harvested, and lysed in sucrose buffer (320 mM sucrose, 3 mM CaCl₂, 2 mM MgOAc, 0.1 mM EDTA, 10 mM DTT, 0.5 mM PMSF, 0.25% NP-40). The nuclei were pelleted by centrifugation (10 min, 1100 g, 4°C) and lysed by incubation with lysis buffer (50 mM HEPES pH 7.8, 3 mM MgCl₂, 300 mM NaCl, 1 mM DTT, 0.1 mM PMSF) in the presence of 15 U/µl of benzonase for 1 hr at 4°C. Before antibody incubation, an input sample was collected and stored at −20°C. Protein G Dynabeads were coupled with 8 µg of anti-Flag antibody (clone M2, #F1804, Sigma-Aldrich) per plate and incubated overnight together with the nuclear extracts. After several washes, immunoprecipitates were eluted in elution buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl) supplemented with 200 µg/ml of 3xFlag peptide (Sigma-Aldrich). As negative control, RH4 wildtype cells were used. For all experiments, at least three biological replicates were performed.

Marques J.G., Gryder B.E., Pavlovic B., Chung Y., Ngo Q.A., Frommelt F., Gstaiger M., Song Y., Benischke K., Laubscher D., Wachtel M., Khan J, & Schäfer B.W. (2020). NuRD subunit CHD4 regulates super-enhancer accessibility in rhabdomyosarcoma and represents a general tumor dependency. eLife, 9, e54993.

+ Open protocol

+ Expand

Cell Cycle Regulation by Rad53 and Chl1

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

Rad53-FLAG expressing cells were grown to 5 × 10⁶ cells/ml in YPAD, synchronized with α-factor for 2 h, washed with YPAD and released in YPAD containing 0.2 M HU (Sigma-Aldrich). Samples were collected at 0, 20, 40, 60, 80, 100 and 120 min after release. Chl1-FLAG and Chl1^K48R-FLAG expressing cells were grown to 5 × 10⁶ cells/ml in YPAD. Chl1-Myc-AID expressing cells were grown to 5 × 10⁶ cells/ml in YPAD and synchronized with α-factor for 2 h. Whole cell extracts were prepared by TCA precipitation and analyzed by SDS–PAGE. Western blotting was performed using an anti-Flag antibody (clone M2; Sigma-Aldrich) or anti-Myc antibody (Santa Cruz [9E10: sc-40]). An anti-PGK1 antibody (22C5D8; Invitrogen) was used to control protein loading.

Batté A., van der Horst S.C., Tittel-Elmer M., Sun S.M., Sharma S., van Leeuwen J., Chabes A, & van Attikum H. (2022). Chl1 helicase controls replication fork progression by regulating dNTP pools. Life Science Alliance, 5(4), e202101153.

+ Open protocol

+ Expand

Chromatin Immunoprecipitation (ChIP) in Yeast

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

ChIP was performed following a previously described protocol (39 (link)). Briefly, chromatin fraction was isolated from yeast cells that were cross-linked with 1% formaldehyde and quenched with 136 mM glycine. Following the sonication step to shear the DNA to ∼750 bp (QSONICA sonicator with a microtip), samples were incubated with anti-FLAG antibody (clone M2 - Sigma) conjugated to Protein G Dynabeads (Life Technologies) overnight at 4°C. After washing, cross-link was reversed by incubation with proteinase K at 42°C for four hours and at 65°C for 12 h. DNA was isolated using MiniElute PCR Purification Kit (Qiagen). qPCR was performed using SensiFAST SYBR no-ROX Mastermix (Bioline) and CFX Connect instrument (Biorad). For each PCR reaction, 10 ng of input or ChIP DNA was used as template. The final concentration of the primer was 0.4 μM each. Cycling conditions were 95°C for 3 min followed by 40 cycles of 95°C for 5 s, 60°C for 10 s, and 72°C for 10 s. Ct values were determined using the CFX Manager software. Ct values for each ChIP samples were first normalized to the ChIP experiment carried out with yeast cells expressing untagged-Sub1 proteins and then divided by the values for the CAN1 locus to calculate the relative fold enrichment. Primers used in the qPCR analysis are listed in the Supplementary Table S1.

Lopez C.R., Singh S., Hambarde S., Griffin W.C., Gao J., Chib S., Yu Y., Ira G., Raney K.D, & Kim N. (2017). Yeast Sub1 and human PC4 are G-quadruplex binding proteins that suppress genome instability at co-transcriptionally formed G4 DNA. Nucleic Acids Research, 45(10), 5850-5862.

+ Open protocol

+ Expand

Western Blot Analysis of FLAG-Tagged Proteins

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

Cells were lysed in lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Nonidet P-40 (v/v), 2 mM EDTA, 2 mM DTT with protease inhibitors). Protein concentrations were normalized by performing a protein estimation assay by BCA and 10 or 20 μg of protein was subjected to SDSPAGE (10% or 4–20%) and transferred to 0.2 μm PVDF, 1 h at 130 volts. After blocking with 5% milk in TTBS-(1X Tris-buffered saline (TBS), 0.05% Tween-20), the membrane was probed with anti-FLAG antibody (Clone M2; Sigma Aldrich) (1/1000). Bound FLAG antibody was detected with goat anti-mouse IgG-HRP (1/2500) using SuperSignal West Dura HRP detection reagents and visualized using an Alphaimager chemiluminescence system (Alpha Innotech).

Rahman T., Nagar A., Duffy E.B., Okuda K., Silverman N, & Harton J.A. (2020). NLRP3 Sensing of Diverse Inflammatory Stimuli Requires Distinct Structural Features. Frontiers in Immunology, 11, 1828.

+ Open protocol

+ Expand

Lung Adenocarcinoma Cell Line Analysis

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

Lung adenocarcinoma cancer cell lines, including 393 P, 344SQ, H1299, and HCC827 cells, were cultured in a humidified atmosphere with 5% CO2 at 37 °C in RPMI 1640 medium supplemented with 10% FBS. Anti-flag antibody (clone M2) was purchased from Sigma. Rabbit anti-GATA3 polyclonal antibody (sc-9009) and normal rabbit IgG were purchased from Santa Cruz. Rabbit anti-Tubulin (2125) antibody was purchased from Cell Signal Technology. Human LH2 and LH3 expression plasmids were gifts from Jonathan Kurie MD (the University of Texas MD Anderson Cancer Center). Human and mouse PLOD2 siRNA were purchased from OriGene and Santa Cruz, respectively. Mouse PLOD3 siRNAs were purchased from Santa Cruz. The ChIP essay kit was purchased from ActiveMotif (#53009).

Liu W., Zhang T., Guo L., Wang Y, & Yang Y. (2018). Lysyl hydroxylases are transcription targets for GATA3 driving lung cancer cell metastasis. Scientific Reports, 8, 11905.

+ Open protocol

+ Expand

Stefin B Expressing iMACs Immunoblot

Cited 1 time

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

+ 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!