3h glutamic acid

Manufactured by PerkinElmer

Sourced in United States

[3H]-glutamic acid is a radioactively labeled form of the amino acid glutamic acid, where the hydrogen atom is replaced with a tritium (3H) isotope. This product is commonly used as a research tool in various scientific applications, such as in vitro and in vivo studies, to investigate the role of glutamic acid in biological processes.

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L-[3,4-3H]-glutamic acid (6 citations) [3H] L-glutamic acid (5 citations)

5 protocols using 3h glutamic acid

Astrocyte Glutamate Uptake Assay

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The uptake of [3H]-glutamic acid was used to determine change in glutamate uptake experienced by astrocytes on soft and stiff surface. The media was removed and replaced by serum free high glucose DMEM containing 50 μM glutamate and 18.5 kBq of [3H]-glutamic acid [Perkin Elmer] which was allowed to incubate at 37 °C of 15 min. Uptake was terminated by removal of working solution and cells washed twice with ice-cold PBS lysed in 10 mM NaOH containing 0.1% Triton X-100. 300 ml of lysate was added to liquid scintillation cocktail [Fisher Scientific] and quantified by counting. The protein content was assayed using Bradford assay [Thermo Scientific Kit 23200]. Results were reported as CPM per mg protein.

Wilson C.L., Hayward S.L, & Kidambi S. (2016). Astrogliosis in a dish: substrate stiffness induces astrogliosis in primary rat astrocytes. RSC advances, 6(41), 34447-34457.

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Radiolabeled Amino Acids for Protein Synthesis

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³H-Leucine (leucine, l-[4,5-³H]), ³H-phenylalanine (phenylalanine, l-[2,3,4,5,6-³H]), ³H-tryptophan (tryptophan, l-[5-3H(N)]), ³H-methionine (l-[methyl-³H]-methionine), ³H-tyrosine (l-[ring-3,5-³H]-tyrosine), ³H-arginine (arginine monohydrochloride l-[2,3,4-³H]-arginine), ³H-lysine (l-[4,5-³H(N)]-lysine), ³H-proline (l-[2,3,4,5-³H]-proline), ³H-serine (l-[³H(G)]-serine), ³H-glycine (glycine, [2-³H]-glycine), ³H-glutamine (l-[3,4-³H(N)]-glutamine), and ³H-glutamic acid (l-[3,4-³H]-glutamic acid) are from PerkinElmer (Waltham, MA).

Bandyopadhyay U., Todorova P., Pavlova N.N., Tada Y., Thompson C.B., Finley L.W, & Overholtzer M. (2022). Leucine retention in lysosomes is regulated by starvation. Proceedings of the National Academy of Sciences of the United States of America, 119(6), e2114912119.

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Radiolabeled Neurotransmitter Uptake Assay

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Non-radiolabeled serotonin and compounds fluoxetine, citalopram, desipramine, paroxetine, cocaine and amphetamine were purchased from Millipore-Sigma (Billerica, MA, USA).
D-PBS was purchased from Invitrogen Life Technologies (Carlsbad, CA, USA). Culture media for cell lines, including Dulbecco’s modified Eagle’s medium (DMEM) with glucose, fetal bovine serum and penicillin/streptomycin were obtained from Thermo Fisher Scientific (Waltham, MA, USA).
Transfection reagent TransIT-LT1 was from Mirus Bio LLC (Madison, WI, USA).
Radiolabeled substrates [³H]-dopamine (53.6 Ci/mmol), [³H]-serotonin (28.2 Ci/mmol), [³H]-norepinephrine (14.9 Ci/mmol), [³H]-glutamic acid (51.1 Ci/mmol), [³H]-glycine (48.7 Ci/mmol) and [³H]-GABA (92.1 Ci/mmol) were purchased from Perkin Elmer (Boston, MA, USA).
Scintillation fluid (Ecolite) was obtained from Thermo Fisher Scientific (Waltham, MA, USA).

Camicia F., Vaca H.R., Guarnaschelli I., Koziol U., Mortensen O.V, & Fontana A.C. (2022). Molecular characterization of the serotonergic transporter from the cestode Echinococcus granulosus: pharmacology and potential role in the nervous system. Parasitology research, 121(5), 1329-1343.

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Glutamate Transporter Regulation Assay

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RX, MnCl₂ and 3,4,5-dimethyl thiazol-2,5-diphenyl tetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO). The cell culture media such as MEM, DMEM and Opti-MEM, and Lipofectamine 2000 were obtained from Invitrogen (Carlsbad, CA). [³H]Glutamic acid was from PerkinElmer. UCPH 101, AG1478, pyrrolidine dithiocarbamate (PDTC), H89, PP2, LY294002 (LY), PD98059 (PD), dihydrokainate (DHK) and G15 were from Tocris Bioscience (Ellisville, MO). GLAST (ab 416) and GLT-1 (ab 58571) antibodies were from Abcam (Cambridge, MA). ERK (sc-135900), phospho-ERK (sc-7383), Akt (sc-55523), phospho-Akt (sc-7985), CREB (sc-186), phospho-CREB (sc-7978), EGFR (sc-120), phospho-EGFR (sc-12357), NF-κB (p65, sc-372 and p50, sc-114), EAAC1 (sc-25658) and β-actin (sc-1616) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). A luciferase reporter assay kit was obtained from Promega (Madison, WI).

Karki P., Webb A., Zerguine A., Choi J., Son D.S, & Lee E. (2014). Mechanism of Raloxifene-induced Upregulation of Glutamate TransporterS in Rat Primary Astrocytes. Glia, 62(8), 1270-1283.

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Aminoacylation Assay for tRNA Glutamylation

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Aminoacylation assays were performed in 20 mM Tris-HCl pH 7.5, 20 mM KCl, 10 mM MgCl₂, 0.1 mg/ml bovine serum albumin (BSA), 4 mM DTT, 4 mM ATP, 20 μM glutamic acid, 0.3 μCi/μl [³H]-glutamic acid (PerkinElmer), and variable amounts of in vitro transcribed human tRNA^Glu(TTC) and MBP-ERS proteins, as indicated in the table and figure legends. Reactions were initiated by addition of MBP-ERS protein to a reaction cocktail containing folded tRNA and other components described above. Reactions were quenched on Whatman 3 MM filter pads presoaked with 5% trichloroacetic acid (TCA). The filter pads were washed three times in excess 5% TCA and one time with 95% ethanol and then dried before counting in a liquid scintillation counter (Beckman Coulter LS 6500). Each assay was performed in triplicate, and kinetic parameters were determined by fitting the data to the Michaelis–Menten equation (see Fig. S5).

Jin D., Wek S.A., Kudlapur N.T., Cantara W.A., Bakhtina M., Wek R.C, & Musier-Forsyth K. (2021). Disease-associated mutations in a bifunctional aminoacyl-tRNA synthetase gene elicit the integrated stress response. The Journal of Biological Chemistry, 297(4), 101203.

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