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Anti flag m2 antibody affinity gel

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The Anti-FLAG M2 antibody affinity gel is a laboratory product designed for the purification and detection of proteins tagged with the FLAG peptide sequence. It consists of an agarose-based resin with covalently coupled Anti-FLAG M2 antibodies, which selectively bind to the FLAG tag fused to the target protein. This gel facilitates the efficient isolation and enrichment of FLAG-tagged proteins from complex samples.

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Lab products found in correlation

Freestyle293 cells, by Thermo Fisher Scientific (8 mentions) Dmrie c reagent, by Thermo Fisher Scientific (1 mentions) Edta free protease inhibitor tablet, by Roche (1 mentions) Amicon ultra centrifugal filter, by Merck Group (1 mentions) Imagequant tl, by GE Healthcare (1 mentions) Flag peptide, by Merck Group (1 mentions)

11 protocols using anti flag m2 antibody affinity gel

1

Engineered Mouse PlexinA1/A2 Sema Domain Protein

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Example 22

A mouse PlexinA1/A2 sema domain chimeric protein gene was designed on the basis of the sequence of NCBI Reference Sequence NP_032907.1 to encode a sequence in which the signal peptide (from the N-terminus to isoleucine at position 24) was replaced with artificial signal peptide HMM+38 (SEQ ID NO: 7), and a sequence from arginine at position 459 to serine at position 510 of the mouse PlexinA2 sema domain protein, the FLAG tag (SEQ ID NO: 5), and the termination codon were added to a sequence after isoleucine at position 458 of the mouse PlexinA1 sema domain protein. This gene was prepared by gene synthesis. The amino acid sequence is shown in SEQ ID NO: 40. The prepared gene was incorporated into an expression vector and then introduced into FreeStyle293 cells from Invitrogen for expression, and the mouse PlexinA1/A2 sema domain protein was purified from the culture supernatant by affinity purification using anti-FLAG M2 antibody affinity gel (Sigma-Aldrich) and gel filtration chromatography.

US10688178B2. AntiPlexin A1 agonist antibody (2020-06-23). OSAKA UNIVERSITY [JP], CHUGAI SEIYAKU KABUSHIKI KAISHA [JP]. Inventors: Atsushi Kumanogoh [JP], Ryusuke Omiya [JP], Hiroyuki Tsunoda [JP], Takeshi Baba [JP], Sachiyo Suzuki [JP], Yuri Teranishi [JP].
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2

Silkworm Pupae Recombinant Protein Production

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Silkworm pupae were used for the expression of recombinant Δ148aa-hACC2 as a bioreactor. To produce recombinant protein in pupae, 10 μg of BmNPV-Δ148aa-hACC2 bacmid DNA was directly injected with DMRIE-C reagent (Invitrogen) into the dorsal of pupae. The injected pupae were reared at 27 °C for 6–7 days, and stored at –80 °C until further analysis. Protein purification was carried out at 4 °C to minimize aggregation and protease activity. Five pupae were homogenized in 10 mL of lysis buffer (50 mM Tris-HCl, 150 mM NaCl, pH 7.4, and 0.1 % TritonX-100) containing an EDTA-free protease inhibitor tablet (Roche, Mannheim, Germany) using a homogenizer (GLH-115, Yamato, Tokyo, Japan). Cell debris was removed by pelleting through centrifugation at 12,000 × g for 30 min. The supernatant was filtered using a 0.45-μm syringe filter and loaded onto a 500-μL of Anti-FLAG M2 antibody Affinity Gel (Sigma-Aldrich, St. Louis, MO, USA) pre-equilibrated with equilibration buffer (50 mM Tris-HCl, 150 mM NaCl, pH 7.4 and 0.02 % TritonX-100). The column was washed with 2.5 mL of equilibration buffer and eluted with elution buffer (100 μg/mL FLAG peptide in 50 mM Tris-HCl and 150 mM NaCl, pH 7.4). The eluted Δ148aa-hACC2 was collected and concentrated using a 100 K Amicon Ultra centrifugal filter (Millipore, Billerica, MA, USA).
Hwang I.W., Makishima Y., Kato T., Park S., Terzic A, & Park E.Y. (2014). Human acetyl-CoA carboxylase 2 expressed in silkworm Bombyx mori exhibits posttranslational biotinylation and phosphorylation. Applied Microbiology and Biotechnology, 98(19), 8201-8209.
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3

Soluble mouse PlexinA1 protein production

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Example 4

Soluble mouse PlexinA1 protein was designed on the basis of the amino acid sequence of NCBI Reference Sequence NP_032907.1 (SEQ ID NO: 52) up to the extracellular domain. The signal peptide (from the N-terminus to isoleucine at position 24) was replaced with artificial signal peptide HMM+38 (SEQ ID NO: 7), and the FLAG tag sequence (SEQ ID NO: 5) was inserted at the C-terminus. The prepared amino acid sequence is shown in (SEQ ID NO: 16). The prepared gene was incorporated into an expression vector and then introduced into FreeStyle293 cells from Invitrogen for expression, and the soluble mouse PlexinA1 protein was purified from the culture supernatant by affinity purification using anti-FLAG M2 antibody affinity gel (Sigma-Aldrich) and gel filtration chromatography.

US10688178B2. AntiPlexin A1 agonist antibody (2020-06-23). OSAKA UNIVERSITY [JP], CHUGAI SEIYAKU KABUSHIKI KAISHA [JP]. Inventors: Atsushi Kumanogoh [JP], Ryusuke Omiya [JP], Hiroyuki Tsunoda [JP], Takeshi Baba [JP], Sachiyo Suzuki [JP], Yuri Teranishi [JP].
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4

Production of Mouse PlexinA1 Sema Domain

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Example 20

A mouse PlexinA1 sema domain protein gene was designed on the basis of the sequence of NCBI Reference Sequence NP 032907.1 to encode a sequence in which the signal peptide (from the N-terminus to isoleucine at position 24) was replaced with artificial signal peptide HMM+38 (SEQ ID NO: 7), and the FLAG tag (SEQ ID NO: 5) and the termination codon were added after serine at position 512. This gene was prepared by gene synthesis. The amino acid sequence is shown in SEQ ID NO: 38. The prepared gene was incorporated into an expression vector and then introduced into FreeStyle293 cells from Invitrogen for expression, and the mouse PlexinA1 sema domain protein was purified from the culture supernatant by affinity purification using anti-FLAG M2 antibody affinity gel (Sigma-Aldrich) and gel filtration chromatography.

US10688178B2. AntiPlexin A1 agonist antibody (2020-06-23). OSAKA UNIVERSITY [JP], CHUGAI SEIYAKU KABUSHIKI KAISHA [JP]. Inventors: Atsushi Kumanogoh [JP], Ryusuke Omiya [JP], Hiroyuki Tsunoda [JP], Takeshi Baba [JP], Sachiyo Suzuki [JP], Yuri Teranishi [JP].
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5

Designing Chimeric Plexin Sema Domains

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Example 22

A mouse PlexinA1/A2 sema domain chimeric protein gene was designed on the basis of the sequence of NCBI Reference Sequence NP_032907.1 to encode a sequence in which the signal peptide (from the N-terminus to isoleucine at position 24) was replaced with artificial signal peptide HMM+38 (SEQ ID NO: 7), and a sequence from arginine at position 459 to serine at position 510 of the mouse PlexinA2 sema domain protein, the FLAG tag (SEQ ID NO: 5), and the termination codon were added to a sequence after isoleucine at position 458 of the mouse PlexinA1 sema domain protein. This gene was prepared by gene synthesis. The amino acid sequence is shown in SEQ ID NO: 40. The prepared gene was incorporated into an expression vector and then introduced into FreeStyle293 cells from Invitrogen for expression, and the mouse PlexinA1/A2 sema domain protein was purified from the culture supernatant by affinity purification using anti-FLAG M2 antibody affinity gel (Sigma-Aldrich) and gel filtration chromatography.

US11576969B2. AntiPlexin A1 agonist antibody (2023-02-14). OSAKA UNIVERSITY [JP], CHUGAI SEIYAKU KABUSHIKI KAISHA [JP]. Inventors: Atsushi Kumanogoh [JP], Ryusuke Omiya [JP], Hiroyuki Tsunoda [JP], Takeshi Baba [JP], Sachiyo Suzuki [JP], Yuri Teranishi [JP].
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6

Recombinant Mouse PlexinA1 Sema Domain

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Example 20

A mouse PlexinA1 sema domain protein gene was designed on the basis of the sequence of NCBI Reference Sequence NP_032907.1 to encode a sequence in which the signal peptide (from the N-terminus to isoleucine at position 24) was replaced with artificial signal peptide HMM+38 (SEQ ID NO: 7), and the FLAG tag (SEQ ID NO: 5) and the termination codon were added after serine at position 512. This gene was prepared by gene synthesis. The amino acid sequence is shown in SEQ ID NO: 38. The prepared gene was incorporated into an expression vector and then introduced into FreeStyle293 cells from Invitrogen for expression, and the mouse PlexinA1 sema domain protein was purified from the culture supernatant by affinity purification using anti-FLAG M2 antibody affinity gel (Sigma-Aldrich) and gel filtration chromatography.

US11576969B2. AntiPlexin A1 agonist antibody (2023-02-14). OSAKA UNIVERSITY [JP], CHUGAI SEIYAKU KABUSHIKI KAISHA [JP]. Inventors: Atsushi Kumanogoh [JP], Ryusuke Omiya [JP], Hiroyuki Tsunoda [JP], Takeshi Baba [JP], Sachiyo Suzuki [JP], Yuri Teranishi [JP].
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7

Purification of Mouse PlexinA2 Sema Domain

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Example 21

A mouse PlexinA2 sema domain protein gene was designed on the basis of the sequence of NCBI Reference Sequence NP 032908.2 (SEQ ID NO: 53) to encode a sequence in which the signal peptide (from the N-terminus to glycine at position 31) was replaced with artificial signal peptide HMM+38 (SEQ ID NO: 7), and the FLAG tag (SEQ ID NO: 5) and the termination codon were added after serine at position 510. This gene was prepared by gene synthesis. The amino acid sequence is shown in SEQ ID NO: 39. The prepared gene was incorporated into an expression vector and then introduced into FreeStyle293 cells from Invitrogen for expression, and the mouse PlexinA2 sema domain protein was purified from the culture supernatant by affinity purification using anti-FLAG M2 antibody affinity gel (Sigma-Aldrich) and gel filtration chromatography.

US10688178B2. AntiPlexin A1 agonist antibody (2020-06-23). OSAKA UNIVERSITY [JP], CHUGAI SEIYAKU KABUSHIKI KAISHA [JP]. Inventors: Atsushi Kumanogoh [JP], Ryusuke Omiya [JP], Hiroyuki Tsunoda [JP], Takeshi Baba [JP], Sachiyo Suzuki [JP], Yuri Teranishi [JP].
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8

Purification of Mouse PlexinA2 Sema Domain

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Example 21

A mouse PlexinA2 sema domain protein gene was designed on the basis of the sequence of NCBI Reference Sequence NP_032908.2 (SEQ ID NO: 53) to encode a sequence in which the signal peptide (from the N-terminus to glycine at position 31) was replaced with artificial signal peptide HMM+38 (SEQ ID NO: 7), and the FLAG tag (SEQ ID NO: 5) and the termination codon were added after serine at position 510. This gene was prepared by gene synthesis. The amino acid sequence is shown in SEQ ID NO: 39. The prepared gene was incorporated into an expression vector and then introduced into FreeStyle293 cells from Invitrogen for expression, and the mouse PlexinA2 sema domain protein was purified from the culture supernatant by affinity purification using anti-FLAG M2 antibody affinity gel (Sigma-Aldrich) and gel filtration chromatography.

US11576969B2. AntiPlexin A1 agonist antibody (2023-02-14). OSAKA UNIVERSITY [JP], CHUGAI SEIYAKU KABUSHIKI KAISHA [JP]. Inventors: Atsushi Kumanogoh [JP], Ryusuke Omiya [JP], Hiroyuki Tsunoda [JP], Takeshi Baba [JP], Sachiyo Suzuki [JP], Yuri Teranishi [JP].
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9

Immunoblotting and Immunoprecipitation Protocols

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IB and immunoprecipitation experiments have been described in detail (Kadowaki et al, 2015 (link)). Cells and iBAT were lysed in lysis buffer (20 mM Tris–HCl, pH 7.5, 150 mM NaCl, 5 mM EGTA, 1% Triton X-100, β-glycerophosphate, and 1% sodium deoxycholate) containing 1 mM phenylmethylsulfonyl fluoride, 1 mM Na3VO4, and 50 mM NaF on ice. The proteins were separated by SDS–PAGE, blotted onto PVDF membranes and blocked in TBS/T containing 5% dry milk. The membranes were incubated with antibodies and detected by an ECL system. The immunoprecipitation experiments were performed using an anti-Flag M2 antibody affinity gel (Sigma-Aldrich). After washing the gels, the immunoprecipitates were detected by IB. Band intensity was measured by ImageQuant TL (GE Healthcare) or ImageJ software (National Institutes of Health). Antibodies are listed in Table S3.
Kato H., Okabe K., Miyake M., Hattori K., Fukaya T., Tanimoto K., Beini S., Mizuguchi M., Torii S., Arakawa S., Ono M., Saito Y., Sugiyama T., Funatsu T., Sato K., Shimizu S., Oyadomari S., Ichijo H., Kadowaki H, & Nishitoh H. (2020). ER-resident sensor PERK is essential for mitochondrial thermogenesis in brown adipose tissue. Life Science Alliance, 3(3), e201900576.
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10

Purification and Analysis of SAMP2-TBP2 Conjugates

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Wild-type and SPS deletion strains (ΔubaA and Δpan1 Δpan2 strains) expressing plasmid pJAM2201 and wt cells expressing pJAM202c were grown to stationary phase in 2 times 1 liter of ATCC 974 medium at 42°C. After cell lysis, equivalent amounts of total cellular proteins, as determined by BCA protein assay, were passed through a Strep-Tactin column as described above. A total of 3 ml of pooled fractions of eluted proteins were subsequently used for immunoprecipitation by anti-Flag M2 antibody affinity gel according to the supplier’s instructions (Sigma). SAMP2-TBP2 conjugates were eluted in Tris buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.4) supplemented with 150 ng·µl−1 Flag peptide (Sigma) and analyzed by immunoblotting.
Fu X., Liu R., Sanchez I., Silva-Sanchez C., Hepowit N.L., Cao S., Chen S, & Maupin-Furlow J. (2016). Ubiquitin-Like Proteasome System Represents a Eukaryotic-Like Pathway for Targeted Proteolysis in Archaea. mBio, 7(3), e00379-16.
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