Dimethyl α ketoglutarate

Manufactured by Merck Group

Sourced in United States, United Kingdom

Dimethyl-α-ketoglutarate is a chemical compound used as a laboratory reagent. It is a diester of α-ketoglutaric acid and methanol. The compound is commonly used in various scientific applications, including biochemical research and analysis.

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Close spelling variants of this product

Dimethyl-α-ketoglutarate (α-KG; Dimethyl-α-Ketoglutarate (DMKG; Dimethyl ketoglutarate α-ketoglutarate

24 protocols using dimethyl α ketoglutarate

Macrophage Activation and Modulation

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At day 7 BMDMs were collected, plated and activated overnight as indicated. Peritoneal cells were obtained by peritoneal lavage with 5ml of PBS. Cells were plated and cultured overnight in complete RPMI 1640 medium (Corning) containing 10mM glucose, 2mM L-glutamine, 100U/ml penicillin/streptomycin, and 10% FBS at 37°C and 5% CO2. Cell were incubated overnight with the following treatments: IL-4 (20ng/mL, Peprotech), AOA (200μM, Sigma), Dimethyl-α-ketoglutarate (1mM, Sigma), EGCG (100μM, Sigma), GSH (10mM, Sigma), L-ornithine (1mM, Sigma), BCATc inhibitor (20μM, Cayman Chemical), Gabapentin (10μg/mL, Sigma), 3NPA (1.68mM, Sigma), Antimycin A (0.1μM, Sigma), Tempol (4mM, EMD Millipore), mitoquinol (200nM, Cayman Chemical), NAC (10mM, Sigma), MDIVI-1 (10μM, Sigma), NSC23766 (50μM, EMD Millipore) or ML141 (10μM, EMD Millipore) (Supplemntary Table. 2).

Merlin J., Ivanov S., Dumont A., Sergushichev A., Gall J., Stunault M., Ayrault M., Vaillant N., Castiglione A., Swain A., Orange F., Gallerand A., Berton T., Martin J.C., Carobbio S., Masson J., Gaisler-Salomon I., Maechler P., Rayport S., Sluimer J.C., Biessen E.A., Guinamard R.R., Gautier E.L., Thorp E.B., Artyomov M.N, & Yvan-Charvet L. (2021). Non-canonical glutamine transamination sustains efferocytosis by coupling redox buffering to oxidative phosphorylation. Nature metabolism, 3(10), 1313-1326.

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Reconstitution of Metabolic Compounds

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Clofibrate and palmitic acid (Sigma-Aldrich, Dorset, UK) were all reconstituted in dimethyl sulfoxide (DMSO). Etomoxir, malonyl-CoA, glucose, dimethyl-α-ketoglutarate, and glutamine (Sigma-Aldrich, Dorset, UK) were reconstituted in E199 medium.

Boodhoo N., Kamble N., Sharif S, & Behboudi S. (2020). Glutaminolysis and Glycolysis Are Essential for Optimal Replication of Marek’s Disease Virus. Journal of Virology, 94(4), e01680-19.

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Comprehensive Reagent Acquisition for Cell Assays

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968 was obtained from Chembridge (San Diego, CA), while BPTES was
a kind gift from Dr. Scott Ulrich (Ithaca College, Ithaca, NY). MDC
and dimethyl-α-ketoglutarate were purchased from Sigma (St.
Louis, MO). T101 and Z-Don were purchased from Zedira GmbH (Darmstadt,
Germany). All of the cell lines used in this study were obtained from
the ATCC (Manassas, VA). Cell culture reagents and SDS-PAGE gels were
obtained from Invitrogen (Carlsbad, CA). Western Lighting Plus ECL
was obtained from PerkinElmer (Waltham, MA). The anti-TG2 cocktail
antibody (MS-300-P) was from Neomarkers (Fremont, CA), and the antivinculin
antibody (V9131) was from Sigma. The HRP-conjugated antirabbit IgG
(70745) was from Cell Signaling (Danvers, MA), and the HRP-conjugated
antimouse IgG (NA931-1 ML) was from GE Healthcare Life Sciences (Pittsburgh,
PA). All of the other reagents were obtained from Thermo Fisher Scientific
(Waltham, MA).

Katt W.P., Antonyak M.A, & Cerione R.A. (2014). Simultaneously Targeting Tissue Transglutaminase and Kidney Type Glutaminase Sensitizes Cancer Cells to Acid Toxicity and Offers New Opportunities for Therapeutic Intervention. Molecular Pharmaceutics, 12(1), 46-55.

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Oxidative Stress-Induced Cell Death Modulators

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ROS were generated by 200 µM hydrogen peroxide (Sigma‐Aldrich, H1009). Cell death inhibitors were used at the following concentrations: ferrostatin‐1 (Fer‐1, 2 µM TargetMol, T6500), liproxstatin‐1 (Lip‐1, 2 µM TargetMol, T2376), necrostatin‐1 (Nec‐1, 10 µM, TargetMol, T1847), Gsk‐872 (10 µM, TargetMol, T4074), Z‐VAD‐FMK (50 µM, TargetMol, T7020). 1S,3R‐RSL3 (TargetMol, T3646) and Erastin (TargetMol, T1765) were used as ferroptosis inducers. For KDM4A/KDM4B inhibition, cells were incubated with NSC 636819 (5 µM, TOCRIS, 5287). For BCAT1 activity inhibition, cells were treated with ERG240 (10 mM, Selleck, E1017) or gabapentin (10 mM, TargetMol, T0702). For BCAA incubation, cells were treated with 0.429, 1.716, or 3.432 mM BCAA mixture (weight ratios of valine: leucine: isoleucine = 1: 2: 1) as previously described.^[³⁵^] For BCKA incubation, cells were treated with 0.429, 1.716, or 3.432 mM BCKA mixture (weight ratios of α‐KIV: α‐KIC: α‐KMV = 1: 2: 1) as previously described.^[³⁹^] For α‐KG incubation, cells were treated with 1, 5, or 10 mM Dimethyl α‐ketoglutarate (Sigma‐Aldrich, 349631) in Figure 4H and 5 mM in further experiments. For glutamate incubation, cells were treated with 0.1, 0.25, or 0.5 mM l‐glutamic acid (Sigma‐Aldrich, G1251). For D‐2‐HG incubation, cells were treated with 5 mM disodium (R)‐2‐hydroxyglutarate (Sigma‐Aldrich, 16859).

Hu G., Cui Z., Chen X., Sun F., Li T., Li C., Zhang L., Guo X., Zhao H., Xia Y., Yan W., Yi W., Fan M., Yang R., Wang S., Tao L, & Zhang F. (2023). Suppressing Mesenchymal Stromal Cell Ferroptosis Via Targeting a Metabolism‐Epigenetics Axis Corrects their Poor Retention and Insufficient Healing Benefits in the Injured Liver Milieu. Advanced Science, 10(13), 2206439.

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NCI-60 Cell Line Anticancer Drug Screen

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The complete NCI-60 panel was obtained from National Cancer Institute (NCI) Anticancer Drug Screen (Bethesda, MD, USA). Pancreatic cell lines were obtained from ATCC (Manassas, VA, USA). All cells were cultivated in RPMI including 2 mM glutamine and 11.11 mM glucose supplemented by 10% FBS and penicillin/streptomycin (GIBCO, Grand Island, NY, USA).
Romidepsin (depsipeptide, NSC 630176) was provided by the Cancer Therapy Evaluation Program, NCI. Tariquidar was provided by the NCI Anticancer Drug Screen. PD-0325901 (MEK inhibitor), MK-2206 (AKT inhibitor) were purchased from ChemieTek (Indianapolis, IN, USA). Glutaminase inhibitor 968 compound was obtained from Millipore (Burlington, MA, USA); oligomycinA, NAC, glutathione, dimethyl-α-ketoglutarate, dimethyl-glutamate, sodium citrate, and sodium acetate were obtained from Sigma-Aldrich (St. Louis, MO, USA); DPI, ML171, and etomoxir was obtained from Selleckchem (Houston, TX). MitoQ was provided by Dan L. Sackett (NIH, NICHD).
Unless otherwise specified, t-tests were performed to evaluate significance of results, and asterisks were added to graphs when the p-value was lower than 0.05.

Basseville A., Violet P.C., Safari M., Sourbier C., Linehan W.M., Robey R.W., Levine M., Sackett D.L, & Bates S.E. (2022). A Histone Deacetylase Inhibitor Induces Acetyl-CoA Depletion Leading to Lethal Metabolic Stress in RAS-Pathway Activated Cells. Cancers, 14(11), 2643.

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Metabolic Regulation of Cell Cycle and Oxidative Stress

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Gelatin, sodium dodecyl sulfate (SDS), dithiothreitol, NaCl, EDTA, heparin, trichloroacetic acid, trypan blue, propidium iodide, RNase, cesium chloride, M199 medium, dimethyl-α-ketoglutarate, aspartate, hydrogen peroxide, glutamine, dialyzed fetal bovine serum, trypsin, 6-diazo-5-oxo-L-norleucine (DON), mercaptoethanol, streptomycin, penicillin, bis-2-(5-phenylacetomido-1,3,4-thiadiazol-2-yl)ethyl sulfide (BPTES), glutamate, ammonium chloride, and diethylamine NONOate (DEA-NO), K₂HPO₄, ATP, NAD, hydrazine, bovine liver glutamate dehydrogenase, and asparagine were from Sigma-Aldrich (St. Louis, MO). Endothelial cell growth factor was from Becton Dickinson Biosciences (Bedford, MA). Rainbow molecular weight markers were from GE Healthcare (Piscataway, NJ). Lipofectamine and 5-(and-6)-chloromethyl-2,7-dichlorodihydrofluorescein diacetate acetyl ester (CM-H₂DCFDA) were from Life Technologies Corporation (Carlsbad, CA). CB-839 and N^G-nitro-L-arginine methyl ester (L-NAME), were from Selleckchem (Houston, TX). Antibodies against GLS1, cyclin D1, cyclin E, cyclin A, p21, p27, p53, and β-actin were from Santa Cruz Biotechnology (Santa Cruz, CA), an antibody against phospho-retinoblastoma protein was from Cell Signaling Technologies (Beverley, MA), and an antibody against HO-1 was from Assay Designs (Ann Arbor, MI). [³H]Thymidine (20 Ci/mmol) was from Perkin Elmer (Boston, MA).

Peyton K.J., Liu X.M., Yu Y., Yates B., Behnammanesh G, & Durante W. (2018). Glutaminase-1 Stimulates the Proliferation, Migration, and Survival of Human Endothelial Cells. Biochemical pharmacology, 156, 204-214.

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Glutamine Deprivation in Mouse ESCs

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For glutamine deprivation experiments in mouse ESCs, cells were
initially plated in standard S/L medium as described above. The following day,
cells were washed with PBS and then cultured in experimental medium containing a
1:1 mix of glutamine-free DMEM (Gibco 11960–051) and glutamine-free
Neurobasal medium (Gibco 21103–049) including 10% dialyzed FBS,
2-mercaptoethanol, and LIF as described above and containing
(“+Q”) or lacking (“−Q”) L-glutamine (2 mM)
as indicated. When indicated, dimethyl-α-ketoglutarate (Sigma 349631)
dissolved in DMSO to 1 M was added to a final concentration of 4 mM. For glucose
and glutamine deprivation experiments, cells were cultured in medium containing
a 1:1 mix of glutamine and glucose-free DMEM (Gibco A14430–01) and
glutamine and glucose-free Neurobasal-A medium (Gibco A24775–01)
including 10% dialyzed FBS and all supplements as described above and containing
or lacking glucose or glutamine as indicated.

Vardhana S.A., Arnold P.K., Rosen B.P., Chen Y., Carey B.W., Huangfu D., Carmona-Fontaine C., Thompson C.B, & Finley L.W. (2019). Glutamine independence is a selectable feature of pluripotent stem cells. Nature metabolism, 1(7), 676-687.

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Glutamine Deprivation in Mouse ESCs

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Purchasing Reagents for Biochemical Assay

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N-acetylcysteine, ammonium chloride, and dimethyl α-ketoglutarate were purchased from Sigma (St. Louis, MO, USA).

Kiesel V.A., Sheeley M.P., Donkin S.S., Wendt M.K., Hursting S.D, & Teegarden D. (2022). Increased Ammonium Toxicity in Response to Exogenous Glutamine in Metastatic Breast Cancer Cells. Metabolites, 12(5), 469.

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Serum Starvation and Metabolic Reactivation

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Cells were seeded in a 10 cm dish to be 70%–80% confluent at the end of the experiment. The day after seeding, cells were starved from serum over night for 16 h. Subsequently, cells were washed with Dulbecco’s phosphate-buffered saline (dPBS) and starved 1 additional hour in Hanks’ Balanced Salt Solution (HBSS) (Thermo scientific, 14025050). Next, cells were reactivated with medium containing dialyzed FBS in presence or absence of 2mM Sodium Pyruvate (Merck Sigma, P8574), in presence or absence of 1mM Dimethyl α-ketoglutarate (Merck Sigma, 349631) and collected after the indicated time points. Time 0’ conditions were collected immediately after the starvation with HBSS.

Rinaldi G., Pranzini E., Van Elsen J., Broekaert D., Funk C.M., Planque M., Doglioni G., Altea-Manzano P., Rossi M., Geldhof V., Teoh S.T., Ross C., Hunter K.W., Lunt S.Y., Grünewald T.G, & Fendt S.M. (2020). In Vivo Evidence for Serine Biosynthesis-Defined Sensitivity of Lung Metastasis, but Not of Primary Breast Tumors, to mTORC1 Inhibition. Molecular cell, 81(2), 386-397.e7.

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