Anti flag m2 affinity gel

Manufactured by Merck Group

Sourced in United States, Germany, China, Japan, United Kingdom, Sao Tome and Principe

The Anti-FLAG M2 affinity gel is a chromatography resin designed for the purification of recombinant proteins tagged with the FLAG peptide sequence. The gel consists of an agarose matrix covalently coupled with the anti-FLAG M2 monoclonal antibody, which specifically binds to the FLAG tag. This allows for the selective capture and purification of FLAG-tagged proteins from complex mixtures.

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Lab products found in correlation

Flag peptide, by Merck Group (151 mentions) 3xflag peptide, by Merck Group (64 mentions) Protease inhibitor cocktail, by Roche (64 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (45 mentions) Protease inhibitor cocktail, by Merck Group (41 mentions) Anti flag, by Merck Group (39 mentions) Complete protease inhibitor cocktail, by Roche (25 mentions) Mg132, by Merck Group (18 mentions) Anti flag antibody, by Merck Group (15 mentions) Pierce bca protein assay kit, by Thermo Fisher Scientific (14 mentions) Pvdf membrane, by Merck Group (14 mentions) Cycloheximide (chx), by Merck Group (12 mentions) Anti ha agarose, by Merck Group (12 mentions) Protease inhibitor, by Roche (12 mentions) Ezview red anti ha affinity gel, by Merck Group (12 mentions) Protease inhibitor, by Merck Group (11 mentions) Phosstop, by Roche (11 mentions) Anti flag m2, by Merck Group (11 mentions) Glutathione sepharose 4b, by GE Healthcare (11 mentions) Benzonase, by Merck Group (11 mentions)

1 413 protocols using anti flag m2 affinity gel

Optimizing Recombinant Protein Purification

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To determine the optimal extraction buffer, the following two extraction buffers were used; 50 mM Tris-HCl, pH 8, 250 mM NaCl with 0.01% Tween 80 (TBS-3) and 50 mM citric buffer, pH 4, 250 mM NaCl with 0.01% Tween 80 (CBS). Frozen agroinfiltrated leaves with TRBO constructs with (prBChE+KDEL) and without KDEL sequence (prBChE) were used to purify prBChE-ER and prBChE from total leaf homogenate. Biomass was ground in liquid nitrogen at a ratio of 1 g:4 ml buffer and the amount of enzymatically active prBChE from total leaf homogenate and total soluble proteins were determined.
Based on the buffer screening experiments, CBS buffer was selected for protein recovery from homogenized leaves. The crude extracts (prBChE-ER and prBChE) were filtered through a 0.22 μm filter (EMD Millipore, Chicago, IL, USA) followed by filtration through a 30 kDa Minimate Tangential Flow Filtration Capsule (Pall Corporation, Ann Arbor, MI, USA). The concentrated retentate was loaded on ANTI-FLAG M2 affinity gel (Sigma–Aldrich, St. Louis, MO, USA) and the target protein was captured and eluted based on the manufacturer’s specifications. Similarly, the recombinant protein from the AWF was filtered, concentrated using a 30 kDa Amicon centrifugal filter units (EMD Millipore, Chicago, IL, USA) and purified using ANTI-FLAG M2 affinity gel (Sigma–Aldrich, St. Louis, MO, USA).

Alkanaimsh S., Karuppanan K., Guerrero A., Tu A.M., Hashimoto B., Hwang M.S., Phu M.L., Arzola L., Lebrilla C.B., Dandekar A.M., Falk B.W., Nandi S., Rodriguez R.L, & McDonald K.A. (2016). Transient Expression of Tetrameric Recombinant Human Butyrylcholinesterase in Nicotiana benthamiana. Frontiers in Plant Science, 7, 743.

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Flag-tagged Tet3 Protein Interaction

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HEK 293 cells harvested 48 hr post-transfection was washed once with ice-cold PBS. All IP steps were carried out at 4°C. HEK293 cell pellets were lysed in ice-cold RIPA buffer [10 mM Tris pH 8.0, 1 mM EDTA, 0.5 mM EGTA, 1% triton, 0.1% sodium deoxycholate, 0.1% SDS, 140 mM NaCl, 0.5 mM DTT, 1× cOmplete Mini EDTA-free protease inhibitor (Roche) and phosphatase inhibitor cocktail (P5726 and P0044, Sigma)] for 20 min with agitation. Lysates were clarified by centrifugation at 16 000 g for 10 min and protein concentrations were determined by Bradford assays (Bio-Rad). For mass spectrometry analysis, lysate was incubated 2 h with pre-washed anti-Flag M2 affinity gel (Sigma). For western analysis, lysate was incubated for 1 h with protein A/G Sepharose (GE healthcare) pre-bound to anti-Flag antibody (Sigma). The IP complexes were washed three times with RIPA buffer, boiled in Laemmli SDS buffer (with 100mM DTT) for 5 min and resolved by SDS-PAGE.
To determine interaction between Flag-tagged Tet3 with H2A.Z in mouse ESCs, cell pellets were lysed in ice-cold TBST buffer [50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, and 1% Triton X-100 1× cOmplete Mini EDTA-free protease inhibitor (Roche)] for 20 min with agitation. IP was performed overnight using mouse IgG agarose (Sigma) or anti-Flag M2 affinity gel and washed four times with TBST buffer.

Rao V.K., Swarnaseetha A., Tham G.H., Lin W.Q., Han B.B., Benoukraf T., Xu G.L, & Ong C.T. (2019). Phosphorylation of Tet3 by cdk5 is critical for robust activation of BRN2 during neuronal differentiation. Nucleic Acids Research, 48(3), 1225-1238.

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PD-L1 Ubiquitination and Interaction Assays

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In immunoprecipitation assays, RBMS1 stable knockdown or pLKO.1 control HEK293T cells were co-transfected with pCDH-PD-L1-3×FLAG and pCDH-HA-ubiquitin for 24 h using Sage LipoPlus reagent (Sage) according to the manufacturer’s instructions. Cells were collected and lysised using IP lysise buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% TritonX-100, PMSF and Cocktail). The cell lysates were subjected to immunoprecipitation using anti-Flag M2 Affinity Gel (Sigma–Aldrich) at 4 ˚C overnight, followed by five times washes with 1 ml of washing buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5% TritonX-100, 10% Glycerol). The affinity elute was analyzed by immunoblotting with anti-FLAG and anti-HA antibodies.
In co-immunoprecipitation assays, HEK293T cells were transfected with pCDH-PD-L1-3×FLAG (or pCDH-Flag empty vector) for 24 h using Sage LipoPlus reagent (Sage) according to the manufacturer’s instructions. Cells were lysised by IP lysise buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% TritonX-100, PMSF and Cocktail). Cell lysates were incubated with anti-Flag M2 Affinity Gel (Sigma Aldrich) at 4 ˚C overnight. Following washes with 1 ml of washing buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5% TritonX-100, 10% Glycerol), eluted protein samples was subjected to western blotting.

Zhang J., Zhang G., Zhang W., Bai L., Wang L., Li T., Yan L., Xu Y., Chen D., Gao W., Gao C., Chen C., Ren M., Jiao Y., Qin H., Sun Y., Zhi L., Qi Y., Zhao J., Liu Q., Liu H, & Wang Y. (2022). Loss of RBMS1 promotes anti-tumor immunity through enabling PD-L1 checkpoint blockade in triple-negative breast cancer. Cell Death and Differentiation, 29(11), 2247-2261.

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Profiling PKCι-Mediated Phosphorylation of Par3 and Par6β

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HEK293T cells transfected with the Flag-tagged PKCι WT or Α120Ε was lysed. After centrifugation, the lysate was mixed with anti-Flag M2 affinity gel (Sigma) at 4 °C for 3 h. The anti-Flag M2 affinity gel-captured PKCι was washed twice with the lysis buffer, once with the kinase assay buffer (50 mM Tris, 100 mM NaCl, 10 mM MgCl₂, 150 μM ATP, pH 8.0), and then released by adding the Flag peptide to a final concentration of 0.1 mg/ml. In each kinase reaction assay, Par3 1–854 (3 μM) and Par6β (3 μM) were mixed with PKCι in 100 μl kinase assay buffer at room temperature for 5 min before centrifugation at 21,130 × g for 10 min. Supernatant was isolated from pellet into a clean tube immediately after centrifugation. The pellet fraction was washed once with kinase assay buffer and thoroughly resuspended with the same buffer to the equal volume as supernatant fraction. Proteins from both fractions were detected by Phos-tag SDS–PAGE with Coomassie blue staining. Phos-tag SDS–PAGE was performed by using a resolving gel containing 50 μM phos-tag acrylamide (NARD, AAL-107) and 0.1 mM MnCl_2. The running buffer consisted of 25 mM Tris, 192 mM glycine, and 0.1% (w/v) SDS.

Liu Z., Yang Y., Gu A., Xu J., Mao Y., Lu H., Hu W., Lei Q.Y., Li Z., Zhang M., Cai Y, & Wen W. (2020). Par complex cluster formation mediated by phase separation. Nature Communications, 11, 2266.

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Immunoprecipitation and Immunoblotting Protocol

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We followed the procedure previously established in our laboratory (Wang et al., 2016 (link); Zhang et al., 2018 (link)). Cells were lysed with lysis buffer, containing 1% of proteinase inhibitor cocktail. For the experiment in which anti-Myc was used to pull-down hOAT4, the lyzed cells were precleared with protein G-agarose beads. Anti-Myc antibody (1:100) was incubated with protein G-agarose beads at 4 °C for 1.5 hours. The precleared cell lysates were then mixed with antibody-bound protein G-agarose beads with rotating at 4 °C overnight. For the experiment in which Anti-Flag M2 affinity gel (Sigma–Aldrich, St. Louis, MO) was used to pull-down Nedd4–2, the precleared cell lysates were mixed with Anti-Flag M2 affinity gel with rotating at 4 °C overnight. Proteins bound to the protein G-agarose beads or Anti-Flag M2 affinity gel were eluted with Urea buffer containing β-mecaptoethanol and examined by immunoblotting with appropriate antibodies.

Wang H., Zhang J, & You G. (2018). The Mechanistic Links between Insulin and Human Organic Anion Transporter 4. International journal of pharmaceutics, 555, 165-174.

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Co-Immunoprecipitation of Cytokine-Receptor Complexes

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For co-IP via ANTI-FLAG M2 affinity gel (Sigma Aldrich), transiently transfected HEK293T cells were lysed in 50 mM Tris-HCl pH 7.5, 1 mM EDTA, 150 mM NaCl, and one complete protease inhibitor mixture tablet/50 ml buffer (Roche Diagnostics) supplemented with 1% Triton X-100 for 1 h on ice. Cytokine-containing lysates were mixed with those containing the full-length receptors. For negative control, cytokine variants were incubated without receptors and vice versa. In total, 30 μl of ANTI-FLAG M2 affinity gel (Sigma Aldrich) was added and incubated overnight at 4 °C under gentle agitation. The samples were washed three times with the abovementioned buffer without 1% Triton X-100, and proteins were eluted by adding 50 μl of 2.5× Laemmli buffer, followed by incubation for 10 min at 95 °C. The resulting supernatants were subjected to Western blot analysis.

Georgy J., Arlt Y., Moll J.M., Ouzin M., Weitz H.T., Gremer L., Willbold D., Grötzinger J., Thives-Kurenbach F., Scheller J, & Floss D.M. (2021). Tryptophan (W) at position 37 of murine IL-12/IL-23 p40 is mandatory for binding to IL-12Rβ1 and subsequent signal transduction. The Journal of Biological Chemistry, 297(5), 101295.

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Purification and Characterization of PRMT5 and MYPT1 Proteins

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PP1cδ, GST-MYPT1^1-1004 and GST-MYPT1^667-1004 were expressed in E. coli and purified as described before51 (link)52 . FT-PRMT5 (Sino Biological) for SPR and phosphorylation assays for MS analysis was free from MEP50 (Fig. S2C). FT-PRMT5wt (FT-PRMT5wt), -PRMT5^T80A and FT-MYPT1 was purified from transfected tsA201 lysates using anti-Flag M2 affinity gel (Sigma Aldrich) applying the manufacturer’s protocol. FT-PRMT5wt and -PRMT5^T80A proteins were bound to anti-Flag M2 affinity gel during kinase and phosphatase assays and in vitro protein arginine methyltransferase assays. FT-PRMT5 was in complex with MEP50 (Fig. S2C). PP1c-free FT-MYPT1 was produced for the execution of phosphatase assays and in vitro protein arginine methyltransferase assays. The FT-MYPT1 bound to the anti-Flag M2 affinity gel was incubated with MYPT1^1-296 peptide13 (link) several times to dissociate the PP1c subunit then FT-MYPT1 was eluted from the beads with 300 μg/ml Flag-peptide (Sigma Aldrich). The lack of PP1c in the FT-MYPT1 preparation was validated by Western blot with anti-PP1c antibody and phosphatase assay with 32P-MLC20 substrate, The purity of all the PP1cδ (Fig. S2D), GST-MYPT1^1-1004 and GST-MYPT1^667-1004 (Fig. S2A) were jugjed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PASGE).

Sipos A., Iván J., Bécsi B., Darula Z., Tamás I., Horváth D., Medzihradszky K.F., Erdődi F, & Lontay B. (2017). Myosin phosphatase and RhoA-activated kinase modulate arginine methylation by the regulation of protein arginine methyltransferase 5 in hepatocellular carcinoma cells. Scientific Reports, 7, 40590.

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Immunoprecipitation and Ubiquitination Assay

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RD or 293T cells were transfected with plasmids from the X-treme GENE transfection reagent (Roche) according to the manufacturer's instructions. After 48 h post-transfection, cells were harvested using lysis buffer (50 mM Tris–HCl, pH 7.4, with 150 mM NaCl, 1 mM EDTA, and 1% Triton-X-100), placed for 30 min on ice, centrifuged at 12 000 × g for 10 min, and subsequently incubated with Anti-FLAG M2 affinity gel (Sigma) for 16 h at 4°C. The Anti-FLAG M2 affinity gel was washed five times with wash buffer (50 mM Tris–HCl, pH 7.4, with 150 mM NaCl). The immunoprecipitation complex was eluted by 2× sample buffer (125 mM Tris–HCl, pH 6.8, with 4% SDS, 20% (v/v) glycerol, and 0.004% bromophenol blue) or in competition with 3× FLAG peptides. Bound proteins were subjected to SDS-PAGE and analyzed by western blotting using specific antibodies. For the in vivo ubiquitination assay, 293T cells were co-transfected with ubiquitin fused with HA tags (HA-Ub), and treated with 20 μM MG132 (Sigma) for 4 h before harvesting. Cells were lysed with lysis buffer containing 5 mM of N-ethylmaleimide (Sigma), and immunoprecipitated with Anti-FLAG M2 affinity gel. The ubiquitinated proteins were detected by western blot using anti-HA antibodies.

Kung Y.A., Hung C.T., Chien K.Y, & Shih S.R. (2016). Control of the negative IRES trans-acting factor KHSRP by ubiquitination. Nucleic Acids Research, 45(1), 271-287.

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Immunoprecipitation of D53 and OsBZR1

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The D53-HA and OsBZR1-FLAG constructs were transformed into the Agrobacterium strain GV3101, then injected into N. benthamiana leaves. Total protein was extracted with lysis buffer (10 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.5 mM EDTA, 2 mM DTT, 0.5% NP-40, protease inhibitor cocktail [P9599; Sigma]), incubated with Anti-FLAG M2 Affinity Gel (A2220; Sigma) for 2-3 h, then washed with buffer (10 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.5 mM EDTA, 2 mM DTT, 0.1% NP-40) three times. Loading buffer (23 SDS) was added to the tubes and proteins were denatured by heating at 95 C for 5 min. D53-HA was detected by anti-HA (28003; NewEast Bioscience), and OsBZR1-FLAG was detected by anti-FLAG antibodies (M20008; Abmart).
The 2-week-old Osbzr1-D-FLAG seedlings were frozen in liquid nitrogen and ground into fine powder. The nuclear protein was extracted and incubated with Anti-FLAG M2 Affinity Gel (Sigma-Aldrich, A2220) for 2-3 h, then washed with buffer (10 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.5 mM EDTA, 0.5% NP-40) 3 times. D53 was detected by anti-D53 (Zhou et al., 2013) , and OsBZR1-FLAG was detected by anti-FLAG antibodies (M20008; Abmart).

Fang Z., Ji Y., Hu J., Guo R., Sun S, & Wang X. (2020). Strigolactones and Brassinosteroids Antagonistically Regulate the Stability of the D53-OsBZR1 Complex to Determine FC1 Expression in Rice Tillering. Molecular plant, 13(4).

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Phosphorylation Site Identification of NIPA

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Phoenix cells were transfected with pCDNA3.1/Zeo Flag hNIPA wild type and MSCV Mig NPM-ALK, Flag was immunoprecipitated using Anti-FLAG-M2 affinity gel (Millipore). For the identification of phosphorylation sites of NIPA, samples were prepared with 1 mM DTT for 5 min at 95 °C and alkylated using 5.5 mM iodacetamide for 30 min at 25 °C. Protein mixtures were separated by SDS-PAGE (4–12% Bis-Tris mini gradient gel) and the 55 kDa region of the gel lanes were cut into 2 equal slices. Gel fractions were in-gel digested using trypsin (Promega, Mannheim, Germany) [33 (link)]. Digests were performed overnight at 37 °C in 0.05 M NH₄HCO₃ (pH 7.5). About 0.1 µg of protease was used for each gel band. Peptides were extracted from the gel slices with ethanol and resulting peptide mixtures were processed on STAGE tips as described [34 (link)]. Sample analysis, data acquisition and processing were performed as previously described [35 (link)].

Gengenbacher A., Müller-Rudorf A., Poggio T., Gräßel L., Dumit V.I., Kreutmair S., Lippert L.J., Duyster J, & Illert A.L. (2019). Proteomic Phosphosite Analysis Identified Crucial NPM-ALK-Mediated NIPA Serine and Threonine Residues. International Journal of Molecular Sciences, 20(16), 4060.

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