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Anti flag conjugated agarose beads

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Anti-Flag-conjugated agarose beads are a type of affinity chromatography resin used for the purification of recombinant proteins that have been engineered to contain a Flag tag. The beads are composed of agarose, a polysaccharide material, which has been coupled with anti-Flag antibodies. This allows for the selective binding and isolation of Flag-tagged proteins from complex mixtures.

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Lab products found in correlation

Protein g conjugated sepharose beads, by Roche (2 mentions) Pierce ip lysis buffer, by Thermo Fisher Scientific (2 mentions) Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions) Glutathione sepharose 4b beads, by GE Healthcare (1 mentions) Protein a g agarose bead, by Santa Cruz Biotechnology (1 mentions) Protein g sepharose, by Roche (1 mentions) Rabbit anti flag, by Merck Group (1 mentions) Rabbit anti ha, by Santa Cruz Biotechnology (1 mentions) Rabbit anti myc, by Santa Cruz Biotechnology (1 mentions) Glutathione beads, by Merck Group (1 mentions) Haloperidol, by Merck Group (1 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions) 3h spiperone, by PerkinElmer (1 mentions) Cell culture media, by Thermo Fisher Scientific (1 mentions) Horseradish peroxidase hrp labeled secondary antibody, by Merck Group (1 mentions) Anti flag m2 antibody, by Merck Group (1 mentions) Phorbol myristate acetate (pma), by Merck Group (1 mentions) Hek293, by American Type Culture Collection (1 mentions) Protease inhibitor tablet, by Roche (1 mentions) Anti flag, by Merck Group (1 mentions)

12 protocols using anti flag conjugated agarose beads

1

Co-immunoprecipitation of FBXW7 and p53

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HEK293T cells were transfected with plasmids encoding GFP-FBXW7-α [28 (link)] and FLAG-p53 (WT) [64 (link)]. 30 hrs after transfection, we used 100 μl of anti-FLAG-conjugated agarose beads (Sigma-Aldrich) to immunoprecipitate p53 or anti-GFP antibody-conjugated agarose beads (MBL International) to immunoprecipitate FBXW7 from whole cell extracts of the HEK293T cells using RIPA buffer (150 mM NaCl) with protease inhibitors. After IP, the beads were washed thoroughly with RIPA buffer. Immunoprecipitated proteins were eluted using 2× SDS loading buffer and then boiled at 95°C for 4 mins. Denatured proteins were subsequently separated on 10% SDS PAGE and immunoblotted against anti-GFP and anti-FLAG antibodies as required after transferring to polyvinylidene fluoride membranes as previously described [65 (link)]. The experiments were repeated on at least three separate occasions.
Li N., Lorenzi F., Kalakouti E., Normatova M., Babaei-Jadidi R., Tomlinson I, & Nateri A.S. (2015). FBXW7-mutated colorectal cancer cells exhibit aberrant expression of phosphorylated-p53 at Serine-15. Oncotarget, 6(11), 9240-9256.
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2

Immunoblotting and Immunoprecipitation Assay

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LR73 cells transfected with the indicated plasmids or BMDMs were incubated with apoptotic cells for 10 min and then lysed using lysis buffer (50 mM Tris (pH 7.6), 150 mM NaCl, 10 mM NaPP, 10 mM NaF, 1 mM Na3VO4, 1% Triton X-100, 10 μg/mL pepstatin, 10 μg/mL leupeptin, 10 μg/mL AEBSF, and 10 μg/mL aprotinin). Proteins in the lysates were separated by SDS-PAGE, transferred onto a nitrocellulose membrane, and detected by appropriate antibodies. For immunoprecipitation, the lysates of cells were incubated with anti-FLAG conjugated-agarose beads (Sigma, A2220), glutathione-sepharose 4B beads (GE healthcare, 17-0756-01, Boston, MA, USA), or protein A/G agarose beads (Santa Cruz, sc-2003) with appropriate antibodies for 2 h. Proteins bound to the beads were detected by immunoblotting. ImageJ was used to quantify band intensities.
Kim D., Moon H., Cho H., Min C., Moon B., Yang S., Lee J., Lee S.A., Park H., Lee D.H., Jeong D., Lee G, & Park D. (2021). Apoptotic Cells Trigger Calcium Entry in Phagocytes by Inducing the Orai1-STIM1 Association. Cells, 10(10), 2702.
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3

Immunoprecipitation and Western Blot Analysis

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Briefly, P19 or HEK293T cells were cultured in DMEM containing 15% heat-inactivated FBS (Hyclone, Waltham, MA, USA), after transfection cells were lysed in modified RIPA buffer for 30 min at 4 °C with rotation. Two-hundred milligram of cell lysate was pre-cleared with Protein G-Sepharose (Roche, Indianapolis, IN, USA) for 1 h, and then was incubated with anti-FLAG conjugated agarose beads (Sigma-Aldrich, St. Louis, MO, USA) overnight at 4 °C with rotation. Agarose beads were washed with TBS buffer and denatured samples were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis electrophoresis. Western blot was performed as described above.
Li T., Zhang X., Jiang K., Liu J, & Liu Z. (2018). Dural effects of oxidative stress on cardiomyogenesis via Gata4 transcription and protein ubiquitination. Cell Death & Disease, 9(2), 246.
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4

Immunoprecipitation and Western Blotting Analysis

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Transfected cells were lysed by rocking at 4 °C for 30 min in Pierce IP lysis buffer (Thermo Fisher Scientific) with a Roche Complete, Mini, EDTA-free protease inhibitor cocktail tablet. The lysates were subjected to immunoprecipitation with anti-Flag-conjugated agarose beads (Sigma) for 2 h at 4 °C. After washing three times with lysis buffer, immunoprecipitates were boiled in 4× NuPAGE LDS sample buffer (Novex), and western blotting was carried out according to the NuPAGE electrophoresis (Novex) protocol with rabbit anti-Flag (1:1000, Sigma), rabbit anti-MYC (1:100, Santa Cruz Biotechnology), and rabbit anti-HA (1:200, Santa Cruz Biotechnology) antibodies.
For tissue preparation, 68 hrs AEL eye discs (n = 40) were collected in cold RIPA lysis buffer (Thermo Fisher Scientific). After centrifuge at 20000 g for 10 min at 4 °C, the supernatant was transferred to a new tube and ready for western blot analysis.
Jin M, & Mardon G. (2016). Distinct Biochemical Activities of Eyes absent During Drosophila Eye Development. Scientific Reports, 6, 23228.
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5

C-MYC and PP2A-C Immunoprecipitation

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Cells were lysed in CHAPS buffer (40mM Hepes pH 7.4, 150mM NaCl, 2mM EDTA, 10mM β-glycerophosphate, 0,3% CHAPS and protease and phosphatase inhibitors). Lysates (1-3mg) were then incubated at 4°C for 30min, following a centrifugation at 4°C for 15min at 13000rpm to remove insoluble debris, equal amounts of proteins were incubated with 1-2μg of monoclonal anti-C-MYC or anti PP2A-C antibodies for at least 2-3 hours. In the case of Flag-PR65A immunoprecipitation, anti-Flag conjugated agarose beads have been used (Sigma Aldrich). Then, protein G conjugated sepharose beads (Roche) were added to anti-C-MYC (C-33) and ati-PP2A-C primary antibodies, and incubated with rotation at 4°C for 1hr. The beads were finally collected by centrifugation and washed four times with the lysis buffer plus 150mM NaCl. Proteins bound to the beads were eluted with 20μl of SDS-PAGE sample buffer and boiled at 95°C for 10min.
Cianfanelli V., Fuoco C., Lorente M., Salazar M., Quondamatteo F., Gherardini P.F., De Zio D., Nazio F., Antonioli M., D’Orazio M., Skobo T., Bordi M., Rohde M., Dalla Valle L., Helmer-Citterich M., Gretzmeier C., Dengjel J., Fimia G.M., Piacentini M., Di Bartolomeo S., Velasco G, & Cecconi F. (2014). AMBRA1 links autophagy to cell proliferation and tumorigenesis by promoting c-MYC dephosphorylation and degradation. Nature cell biology, 17(1), 20-30.
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6

Ubiquitin-Mediated Protein Interactions

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293T cells were transfected with an HA-tagged ubiquitin plasmid and other plasmids (flag-klf4 and/or his-plk1). Twenty-four to 48 h after transfection, the cells were lysed in denaturing buffer (1% SDS, 50 mM Tris pH 8.0, 10 mM EDTA). After incubation at 4°C for 10 min, the lysate was sonicated and diluted tenfold with buffer (0.01% SDS, 1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris pH 8.0, 167 mM NaCl) and sonicated again. Then, the cells were incubated with anti-Flag-conjugated agarose beads (Sigma-Aldrich, mouse antibody) for 2H at 4°C. The immunoprecipitates were washed five times with 1×PBS before being resolved by SDS-PAGE and immunoblotted with the indicated antibodies.
Mai J., Zhong Z.Y., Guo G.F., Chen X.X., Xiang Y.Q., Li X., Zhang H.L., Chen Y.H., Xu X.L., Wu R.Y., Yu Y., Li Z.L., Peng X.D., Huang Y., Zhou L.H., Feng G.K., Guo X., Deng R, & Zhu X.F. (2019). Polo-Like Kinase 1 phosphorylates and stabilizes KLF4 to promote tumorigenesis in nasopharyngeal carcinoma. Theranostics, 9(12), 3541-3554.
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7

Dopamine receptor signaling study in HEK293 cells

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Human embryonic kidney cells (HEK-293) were obtained from the American Type Culture Collection (Rockville, MD, USA). Cell culture media and fetal bovine serum were obtained from Life Technologies, Inc. (Carlsbad, CA, USA). Dopamine (DA), (-) quinpirole, haloperidol, PMA, anti-Flag M2 antibodies, anti-Flag-conjugated agarose beads, antibodies for green fluorescence protein (GFP), horseradish peroxidase (HRP)-labeled secondary antibodies, and glutathione beads were purchased from Sigma/Aldrich Chemical (St Louis, MO, USA). [3H]-Sulpride (87 Ci/mmol) and [3H]-spiperone (85.7 Ci/mmol) were purchased from PerkinElmer Life Sciences (Boston, MA, USA).
Zheng M., Zhang X., Min C., Choi B.G., Oh I.J, & Kim K.M. (2016). Functional Regulation of Dopamine D3 Receptor through Interaction with PICK1. Biomolecules & Therapeutics, 24(5), 475-481.
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8

NFATc1 Ubiquitination by TRIP12 Regulation

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Plasmids ubiquitin-HA, NFATc1-flag, and TRIP12-WT-myc, or TRIP12-K1136R/W-myc, or TRIP12-ΔHECT-myc were co-transfected into HEK293T cells. The HEK293T cells were treated with 20 µM MG-132 for 6 hours before harvest. NFATc1 was purified by co-immunoprecipitation using anti-flag-conjugated agarose beads (Sigma), followed by western blot analysis. To examine the effect of WARS on ubiquitination of NFATc1, siWARS were co-transfected with ubiquitin-HA, NFATc1-flag, and TRIP12-WT-myc in HEK293T cells. Alternatively, WARS were reintroduced into HEK293T-WARS-KO cells by co-transfection with ubiquitin-HA, NFATc1-flag, and TRIP12-WT-myc.
Qin R., Zhao C., Wang C.J., Xu W., Zhao J.Y., Lin Y., Yuan Y.Y., Lin P.C., Li Y., Zhao S, & Huang Y. (2021). Tryptophan potentiates CD8+ T cells against cancer cells by TRIP12 tryptophanylation and surface PD-1 downregulation. Journal for Immunotherapy of Cancer, 9(7), e002840.
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9

Immunoprecipitation of Transfected Proteins

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Cells were transiently transfected with the indicated plasmids. Twenty-four hours after transfection, cells were harvested with RIPA buffer and briefly sonicated at 4°C. Lysates were immunoprecipitated with anti-myc or anti-flag conjugated agarose beads (Sigma). Precipitates were analyzed on SDS–polyacrylamide gels.
Li G., Ci W., Karmakar S., Chen K., Dhar R., Fan Z., Guo Z., Zhang J., Ke Y., Wang L., Zhuang M., Hu S., Li X., Zhou L., Li X., Calabrese M.F., Watson E.R., Prasad S.M., Rinker-Schaeffer C., Eggener S.E., Stricker T., Tian Y., Schulman B.A., Liu J, & White K.P. (2014). SPOP promotes tumorigenesis by acting as a key regulatory hub in kidney cancer. Cancer cell, 25(4), 455-468.
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10

Purification of NRARP protein complex

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Jurkat cells were transduced with a retrovirus encoding NRARP with an N-terminal tandem HA-FLAG tag and a cassette encoding the interleukin-2 receptor (IL2R) after an internal ribosomal entry site (IRES) (pOZ-FH-N) (38 (link)). Stable cell lines were generated by magnetic sorting for IL2R-positive cells. For proteomic studies, the resulting cell line was grown in RPMI with 10% bovine growth serum to a density of 3×106 cells/ml. Cells were harvested by centrifugation, and lysed with a Dounce homogenizer. The resulting lysate was immunoprecipitated using anti-FLAG-conjugated agarose beads (Sigma) in 50 mM Tris buffer, pH 7.5, containing 150 mM NaCl, 1 mM EDTA, 0.5% NP40, 2 mM TCEP and 10% glycerol, supplemented with EDTA-free protease inhibitor tablets (Roche). The beads were washed three times and the immunoprecipitated protein was eluted with Flag peptide. The eluate was then re-immunoprecipitated with anti-HA-conjugated agarose beads and eluted from the beads with HA peptide for mass spectrometry analysis.
Jarrett S.M., Seegar T.C., Andrews M., Adelmant G., Marto J.A., Aster J.C, & Blacklow S.C. (2019). Extension of the Notch intracellular domain Ankyrin Repeat Stack by NRARP Promotes Feedback Inhibition of Notch Signaling. Science signaling, 12(606), eaay2369.
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