Methionine free medium

Manufactured by Thermo Fisher Scientific

Sourced in United States

Methionine-free medium is a cell culture medium that lacks the amino acid methionine. It is designed for specific applications that require the removal or depletion of methionine from the cell culture environment.

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Lab products found in correlation

Cycloheximide (chx), by Abcam (1 mentions) Click it hpg alexa fluor 488 protein synthesis assay kit, by Thermo Fisher Scientific (1 mentions) Np40 lysis buffer, by Thermo Fisher Scientific (1 mentions) Irdye 800cw streptavidin, by LI COR (1 mentions) Click go protein reaction buffer kit, by Vector Laboratories (1 mentions) Protein a magnetic beads, by Bio-Rad (1 mentions) Biotin alkyne, by Vector Laboratories (1 mentions) L azidohomoalanine, by Vector Laboratories (1 mentions) Odyssey blocking buffer, by LI COR (1 mentions) Image studio, by LI COR (1 mentions) Alexa fluor conjugated antibody, by Thermo Fisher Scientific (1 mentions) Click it cell reaction kit, by Thermo Fisher Scientific (1 mentions) Dapi was, by Vector Laboratories (1 mentions) Rnaseout, by Thermo Fisher Scientific (1 mentions) Tcs sp5, by Leica camera (1 mentions) Fetal bovine serum (fbs), by Merck Group (1 mentions) Cycloheximide (chx), by Merck Group (1 mentions)

4 protocols using methionine free medium

Measurement of Nascent Protein Synthesis in GBM Cells

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GBM cells were labelled for nascent protein synthesis using a Click-iT HPG Alexa Fluor 488 Protein Synthesis Assay Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Briefly, spheres were dissociated into single cells and incubated in methionine-free medium (Thermo Fisher Scientific) for 30 min and subsequently incubated with HPG for 1 h. During pre-incubation, the control samples were treated with 100 μg ml⁻¹ cycloheximide (Abcam) to inhibit de novo protein synthesis. The cells were fixed with 4% PFA in PBS for 15 min and permeabilized with 0.25% Triton X-100 in PBS for 20 min, followed by two washes with 3% bovine serum albumin. Cells were then stained using Alexa Fluor azide. After extensive washing, the cells were analysed by flow cytometry or imaged using a fluorescence microscope.

Piazza P.V., Barrot M., Rougé-Pont F., Marinelli M., Maccari S., Abrous D.N., Simon H, & Le Moal M. (1996). Suppression of glucocorticoid secretion and antipsychotic drugs have similar effects on the mesolimbic dopaminergic transmission. Proceedings of the National Academy of Sciences of the United States of America, 93(26), 15445-15450.

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Quantifying ZEB1 Nascent Translation

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One day after seeding 300, 000 cells/well, cells were infected with AdSLFN12 or AdCMV. Sixty hours later, the medium was replaced with methionine-free medium (Thermo Fisher, #21013024) for two hours. Cells were incubated with 50μM L-Azidohomoalanine (AHA) (Clickchemistrytools, Scottsdale, AZ, #106625) for 8 hours and lysed in NP-40 lysis buffer (Thermo Fisher, #FNN0021) with 1%SDS. AHA was detected using click chemistry with 50μM Biotin Alkyne (Clickchemistrytools, #1266–5) with Click-&-Go^™ Protein Reaction Buffer Kit (Clickchemistrytools, #1262) per manufacturer’s protocol. After click chemistry, ZEB1 was immunoprecipitated using rabbit anti-ZEB1 and protein-A magnetic beads (Bio-Rad Laboratories, #161–4013). Proteins were resolved by 10% SDS-PAGE, transferred to 0.45μm nitrocellulose membrane, and blocked with Odyssey blocking buffer (LI-COR, Lincoln, NE, #927–50010). Biotin was labeled with IRDye 800CW Streptavidin per manufacturer’s protocol (LI-COR, #926–32230) and ZEB1 was labeled with IRDye 680LT Donkey anti-Rabbit IgG Secondary Antibody. Images were acquired using a LI-COR-Clx and analyzed using Image Studio (LI-COR).

Al-Marsoummi S., Vomhof-DeKrey E, & Basson M.D. (2019). Schlafen12 Reduces the Aggressiveness of Triple Negative Breast Cancer through Post-Transcriptional Regulation of ZEB1 That Drives Stem Cell Differentiation. Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology, 53(6), 999-1014.

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Immunofluorescence Analysis of Oocyte Development

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Oocytes were fixed in 4% paraformaldehyde (PFA) in PBS for 30 min, permeabilized for 15 min in PBS with 0.1% Triton X-100 and incubated overnight at 4 °C with primary antibodies (1:100) against 4E-BP1(T70), 4E-BP1(T37/46), S6K(T389; Cell Signaling Technology), RPS6 (Santa Cruz), LMN A/C or α-tubulin (Sigma). After washing, the oocytes were incubated for 1 h at room temperature with an Alexa Fluor conjugated antibodies (1:250; Molecular Probes). RNaseOut (500 U ml⁻¹; Invitrogen) was used in all the buffers. For nascent protein synthesis specific stage (GV-0 h, NEBD-2 h, pro-MI-7 h, MII-12 h) oocytes were cultured in the methionine-free medium (Gibco) supplemented with 1% dialyzed fetal bovine serum (10,000 MW; Sigma) and 50 μM HPG for 30 min77 (link). HPG was detected by using Click-iT Cell Reaction Kit (Life Technologies). Chromosome spreads from mouse oocytes were prepared as previously described78 (link). ER was detected by 1 μM ER-Tracker (Green dye and Blue-White DPX dye for double staining; Molecular Probes) in M16 for 1 h. DAPI was used for chromosome staining (Vectashield). Nucleic acids were labelled by 50 nM SYTO14 (Molecular Probes) in M16 for 20 min then fixed by PFA and imaged. Samples were visualized using an inverted confocal microscope in 16 bit depth (TCS SP5; Leica). Images were assembled in Photoshop CS3 and quantified by Image J software.

Susor A., Jansova D., Cerna R., Danylevska A., Anger M., Toralova T., Malik R., Supolikova J., Cook M.S., Oh J.S, & Kubelka M. (2015). Temporal and spatial regulation of translation in the mammalian oocyte via the mTOR–eIF4F pathway. Nature Communications, 6, 6078.

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Quantifying Protein Synthesis in U2OS Cells

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Following in vitro transcribed mRNA transfection, U2OS cells were incubated for 30 min with methionine-free medium (Gibco, USA) with 1% l-glutamine (Sigma, Biological Industries, 1639), and were fixed in 4% PFA. Nascent proteins were marked using the Click-iT HPG Alexa Flour 594 protein synthesis assay kit (Invitrogen, C10429), according to the manufacturer’s instructions, and were observed using fluorescence microscopy. Cycloheximide (100 μg/ml, Sigma, C4859) was applied 2 h before fixation.

Angel M., Fleshler E., Atrash M.K., Kinor N., Benichou J.I, & Shav-Tal Y. (2024). Nuclear RNA-related processes modulate the assembly of cytoplasmic RNA granules. Nucleic Acids Research, 52(9), 5356-5375.

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