Methyl 14c choline

Manufactured by PerkinElmer

Sourced in Spain

[methyl-14C]-choline is a radioactively labeled compound used for research purposes. It contains a carbon-14 label on the methyl group of the choline molecule. This product is intended for use in scientific investigations, but a detailed description of its core function is not available while maintaining an unbiased and factual approach.

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Lab products found in correlation

Fla 7000, by Fujifilm (2 mentions) Trans blot turbo mini pvdf, by Bio-Rad (2 mentions) Horseradish peroxidase hrp linked secondary iggs, by Cell Signaling Technology (2 mentions) Mini protean tgx stain free protein gel, by Bio-Rad (2 mentions) Protease inhibitor cocktail, by Merck Group (2 mentions) Clarity western ecl substrate, by Bio-Rad (2 mentions) Hyclone fetal bovine serum, by Thermo Fisher Scientific (1 mentions) 1 14c oleic acid, by PerkinElmer (1 mentions) 3h acetic acid, by PerkinElmer (1 mentions) 9 10 3h palmitic acid, by PerkinElmer (1 mentions) Trichloroacetic acid, by Thermo Fisher Scientific (1 mentions) Hiprep 26 60 sephacryl 100 hr column, by GE Healthcare (1 mentions) Escherichia coli bl21 star de3 cells, by Thermo Fisher Scientific (1 mentions) Ni nta agarose bead, by Qiagen (1 mentions) Pet28a vector, by Thermo Fisher Scientific (1 mentions) Rpmi 1640, by Biowest (1 mentions) Phosphatidylcholine assay kit, by Merck Group (1 mentions) Mitochondrial membrane potential kit, by Merck Group (1 mentions) Tlc plate, by Merck Group (1 mentions) Phospho ampkα, by Cell Signaling Technology (1 mentions)

5 protocols using methyl 14c choline

Measurement of Choline Incorporation

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Cells in 6-well plates (1.0 × 10⁶ cells/well) were incubated for 24 h then treated with JAS239 or MN58b. After 1 h, cells were spiked for 1 h with 0.5 μCi/mL [methyl-¹⁴C]-choline (PerkinElmer) and fixed with trichloroacetic acid. Aqueous cellular extracts were separated by thin layer chromatography (TLC) and the ¹⁴C-PC production measured by autoradiography on a Fujifilm FLA-7000 using previously described methods [4 (link), 23 (link)].

Arlauckas S.P., Kumar M., Popov A.V., Poptani H, & Delikatny E.J. (2017). Near infrared fluorescent imaging of choline kinase alpha expression and inhibition in breast tumors. Oncotarget, 8(10), 16518-16530.

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Radiolabeled Lipid Uptake and Metabolism

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[³²P]inorganic phosphate (³²P_i; 9,000 Ci/mmol), [9,10-³H]palmitic acid (43 Ci/mmole), [1–¹⁴C]oleic acid (54.6 mCi/mmol), and [³H]acetic acid (100 mCi/mmol) and [methyl-¹⁴C]choline (50 mCi/mmol) were obtained from Perkin Elmer (Boston, MA). Lipid standards were purchased from Avanti Polar Lipids. Silica gel G and silica gel 60 thin-layer chromatography plates (EMD), Ham's F12 and DMEM medium (Cellgro or Gibco), fetal bovine serum (HyClone) and tissue culture dishes were obtained from ThermoFisher Scientific. All other reagents, unless otherwise specified, were purchased from Aldrich/Sigma.

Zoeller R.A, & Geoghegan-Barek K. (2019). A cell-based high-throughput screen identifies tyrphostin AG 879 as an inhibitor of animal cell phospholipid and fatty acid biosynthesis. Biochemistry and Biophysics Reports, 18, 100621.

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ChoK Inhibition Assay for Cell Lines

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For each cell line (F98, 9L and 9L-EGFRviii), 5×10⁵ cells/well were seeded in a 6-well plate and incubated for 24 h at 37°C. The exponentially growing cells were pulsed for 1 h with 0.5 μCi/mL of [methyl-¹⁴C]-choline (Perkin Elmer, Shelton, CT) per well at 37°C followed by the addition of varying concentrations of MN58b, which was synthesized in house as previously described (13 ). After 2 h treatment, the medium was removed and cells were washed twice with ice-cold PBS and fixed in 16% ice-cold trichloroacetic acid (Fisher Scientific, Fair Lawn, NJ). ChoK inhibition was probed at 2 h because this time point has been found previously to be a time prior to significant loss in cell viability, thus providing a more accurate measurement of ChoK activity (13 ). Each sample was washed 3x in diethyl ether, lyophilized, and resuspended in water for thin layer chromatography (TLC) separation using a solvent system of NaCl/CH₃OH/NH₄OH; 50:70:0.5. The TLC plates were analyzed by autoradiography using a Fujifilm FLA-7000 (Tokyo, Japan) to detect radioactivity.

Kumar M., Arlauckas S.P., Saksena S., Verma G., Ittyerah R., Pickup S., Popov A.V., Delikatny E.J, & Poptani H. (2015). Magnetic resonance spectroscopy for detection of choline kinase inhibition in the treatment of brain tumors. Molecular cancer therapeutics, 14(4), 899-908.

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Choline Metabolism Enzyme Purification

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Foetal Bovine Serum (FBS), Eagle’s Minimum Essential Medium (MEM) and RPMI-1640 (Roswell Park Memorial Institute 1640) were obtained from Biowest (Nuaillé, France). Thin Layer Chromatography (TLC) plates and protease inhibitor cocktail were from Sigma-Aldrich (Madrid, Spain). [Methyl-¹⁴C]choline was from Perkin Elmer (Madrid, Spain). Mini-PROTEAN^® TGX Stain-Free Protein Gels, Trans-Blot Turbo Mini PVDF and Clarity Western ECL substrate were from Bio-Rad Laboratories, Inc. (Madrid, Spain). Monoclonal anti-human primary antibodies ChoKα (sc-23382) and polyclonal β-actin were from Santa Cruz Biotechnology, Inc. (Heidelberg, Germany). Rabbit polyclonal SLC44A1/CTL1 antibody (ab110767) was from Abcam (Cambridge, MA, USA). Horseradish peroxidase (HRP)-linked secondary IgGs were from Cell Signaling Technology (Danvers, MA, USA). The pET-28a vector and Escherichia coli BL21 (DE3) Star cells were from Invitrogen (Carlsbad, CA, USA), N-terminal 6x His-tag was purchased from Genescript (Piscataway, NJ, USA), Ni-NTA agarose beads were from Qiagen (Venlo, The Netherlands), and HiPrep 26/60 Sephacryl 100 HR column was from GE Healthcare (Little Chalfont, Buckinghamshire, UK). All other reagents were of analytical grade.

García-Molina P., Sola-Leyva A., Luque-Navarro P.M., Laso A., Ríos-Marco P., Ríos A., Lanari D., Torretta A., Parisini E., López-Cara L.C., Marco C, & Carrasco-Jiménez M.P. (2022). Anticancer Activity of the Choline Kinase Inhibitor PL48 Is Due to Selective Disruption of Choline Metabolism and Transport Systems in Cancer Cell Lines. Pharmaceutics, 14(2), 426.

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Choline Metabolism Regulation in Cell Death

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FBS and MEM were obtained from Biowest (Nuaillé, France). The TLC plates, protease-inhibitor cocktail, fluorimetric intracellular ROS kit, mitochondrial membrane potential kit, phosphatidylcholine assay kit, and caspase 3 assay kit were from Sigma-Aldrich (Madrid, Spain). The Muse Annexin V & Dead Cell Assay was from Merck Chemicals & Life Science (Madrid, Spain). [Methyl-¹⁴C]choline, [1,2-¹⁴C]acetic acid and [2-³H]glycerol were from Perkin Elmer (Madrid, Spain). Mini-PROTEAN® TGX Stain-Free™ Protein Gels, Trans-Blot® Turbo™ Mini PVDF and Clarity™ Western ECL substrate were from Bio-Rad Laboratories, Inc. (Madrid, Spain). Monoclonal anti-human primary antibody ChoKα and polyclonal β-actin were from Santa Cruz Biotechnology, Inc. (Heidelberg, Germany). Monoclonal anti-human primary antibodies (p62, AMPKα, Phospho-AMPKα, CHOP, IRE1α), polyclonal anti-human primary antibodies (LC3A/B and Beclin-1) and horseradish peroxidase (HRP)-linked secondary IgGs were from Cell Signaling Technology (Danvers, MA, USA). Polyclonal anti-human antibody, SREBP-2, and monoclonal anti-human antibodies LDLR and HMGCR were from Abcam (Cambridge, MA, USA).

Sola-Leyva A., López-Cara L.C., Ríos-Marco P., Ríos A., Marco C, & Carrasco-Jiménez M.P. (2019). Choline kinase inhibitors EB-3D and EB-3P interferes with lipid homeostasis in HepG2 cells. Scientific Reports, 9, 5109.

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