Cells were washed twice and serum starved for 16–18 h prior to EGF stimulation. Cells were stimulated with EGF in serum-free DMEM low glucose without L-ARG, L-LEU, L-LYS, sodium pyruvate and phenol red (Sigma Aldrich) for 1 h: [3H]Leucine (PerkinElmer, Waltham, MA, USA) was added at the same time as EGF to a final concentration of 5 μCi/ml. Cells were washed three times in ice-cold PBS, lysed using RIPA buffer followed by incubating cells with 10 % trichloroacetic acid (TCA) for 10 minutes to precipitate proteins. Pellets were washed three times in 10 % TCA. Pellets were resuspended in 50 nM NaOH with 1 % Triton X-100 at 65 °C for 30 minutes or until the pellet dissolved. The radioactivity of samples was assessed by measuring the scintillation count using the β-scintillation counter. The results were normalised for protein content using bicinchoninic acid (BCA) analysis.
L lys
Manufactured by Merck Group
Sourced in United States
L-Lys is a laboratory reagent used in various biochemical and analytical applications. It is a pure form of the amino acid lysine, which is a fundamental building block of proteins. L-Lys is commonly utilized in research and testing environments to support protein synthesis, enzyme activity, and other cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
L arg, by Merck Group (4 mentions)
L leu, by Merck Group (2 mentions)
L pro, by Merck Group (2 mentions)
L asp, by Merck Group (2 mentions)
L his, by Merck Group (2 mentions)
Phenol red, by Merck Group (1 mentions)
3h leucine, by PerkinElmer (1 mentions)
B27 supplement, by Thermo Fisher Scientific (1 mentions)
Stempro 34 sfm, by Thermo Fisher Scientific (1 mentions)
Stempro supplement, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Sodium pyruvate, by Merck Group (1 mentions)
Tyramine hydrochloride, by Merck Group (1 mentions)
Tryptamine hydrochloride, by Merck Group (1 mentions)
Putrescine dihydrochloride, by Merck Group (1 mentions)
Cadaverine dihydrochloride, by Merck Group (1 mentions)
Histamine dihydrochloride, by Merck Group (1 mentions)
L phe, by Merck Group (1 mentions)
L tyr, by Merck Group (1 mentions)
6 protocols using l lys
1
Quantifying Protein Synthesis via EGF Stimulation
2
Serum-free Culture of Germ Cells
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We attempted to establish a serum-free medium to assess the effect of FBS and determine a potential alternative. The serum-free medium comprised StemPro-34 SFM (Invitrogen, Carlsbad, CA, USA) supplemented with StemPro Supplement (Invitrogen, USA). The FBS was replaced with 0.5% (w/v) bovine serum albumin (BSA; Sigma-Aldrich, St. Louis, MO, USA), 2 mM L-glutamine (Sigma-Aldrich, St Louis, MO, USA), 0.5% (v/v) B27 Supplement (Gibco, USA), 20 μg/mL L-Lys, 20 μg/mL L-Pro, 20 μg/mL L-Asp, and 10 mM/L sodium pyruvate (Sigma-Aldrich, St Louis, MO, USA). We also added 25 mM HEPES, 50 μg/mL ampicillin, 200 U/mL penicillin and 20 μg/mL streptomycin (pH 8.0). Purified germ cells were cultured at 21 °C with serum-free medium and optimal growth factors. The mitotic activity of germ cells and somatic cells was investigated by BrdU and immunocytochemical assay after 10 d culture.
3
Biogenic Amine and Amino Acid Analysis
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BA standards (tyramine hydrochloride, 2-phenylethylamine hydrochloride, putrescine dihydrochloride, cadaverine dihydrochloride, histamine dihydrochloride, tryptamine hydrochloride, spermidine trihydrochloride) and amino acid standards (L-Asp, L-Glu, L-Ser, L-Gly, L-His, L-Thr, L-Arg, γ-aminobutyric acid [GABA], L-Ala, L-Pro, L-Tyr, L-Val, L-Met, L-Iso, L-Leu, L-Phe, L-Trp, and L-Lys) were all purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Dansyl chloride, perchloric acid, sodium hydrogen carbonate, potassium dichromate, and silver nitrate were purchased from Daejung Chemical Co. (Siheung, Korea). Distilled water, acetone, and acetonitrile (HPLC grade) were purchased from Tedia Co. (Fairfield, OH, USA).
4
Substrate-dependent Amino Acid Transport Assay
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Oocyte recordings were performed in ND100 medium (100 mM NaCl, 2 mM KCl, 1 mM MgCl2, and 1.8 mM CaCl2) buffered with 5 mM 2-(N-morpholino)ethanesulfonic acid (MES)-NaOH to pH 5.00, unless stated otherwise. L-Arg, L-Lys, or L-His (Sigma-Aldrich) were added as monohydrochloride salts. For substrate-free solutions, the amino acid was replaced by NMDG hydrochloride (Sigma-Aldrich) to keep the chloride concentration and osmolarity unchanged. The maximum concentration of NMDG used (20 mM) had no effect on PQCL2 and endogenous currents. For patch-clamp recordings in HEK293T cells, the substrate-free external solution contained the following: 130 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 20 mM glucose, 20 mM NMDG-Cl, and 20 mM MES adjusted to pH 5.00 with NaOH or HCl (osmolarity: 315 to 330 mOsm). For substrate application, NMDG was replaced by 20 mM Lys, Arg, or His. The micropipette solution contained the following: 130 mM CsCl, 10 mM ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), 2 mM CaCl2, 2 mM MgCl2, and 10 mM Hepes adjusted to pH 7.35 to 7.40 with KOH or HCl, supplemented with 20 mM NMDG, Arg, or Lys (osmolarity: 295 to 305 mOsm). L-[2,3,4-3H]arginine monhydrochloride (specific activity: 40 to 50 Ci/mmol) was from Perkin-Elmer.
5
Synthesis of Labeled Biomolecules Protocols
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For use in syntheses, 2,5-dihydro-2,5-dimethoxyfuran (97%, mixture of cis and trans, DMDHF), 2,5-dihydro-2,5-dimethoxy-2-methylfuran (97%, mixture of cis and trans, MDMDHF), l -Lys (≥98%), sodium hydroxide, sodium bicarbonate, acetic acid anhydride, GSH (≥98%), [Gly-13C2,15N]-GSH trifluoroacetate (15N ≥ 98%, 13C ≥ 99%, CP ≥ 95%) and N-α-acetyllysine were purchased from Sigma-Aldrich (St. Louis, USA), whereas acetic acid (99%) was from Avantor Performance Materials B.V. (Radnor, USA) and [13C6,15N2]-Lys x 2HCl (13C6 ≥ 99%; 15N2 ≥ 99%, CP ≥ 98%) was from Carl Roth (Karlsruhe, Germany). For preparative purposes, HPLC-grade formic acid and acetonitrile were purchased from Sigma-Aldrich (St. Louis, MO, USA) and Fisher (Schwerte, Germany), respectively, whereas MS-grade solvents formic acid and acetonitrile were purchased from Carl Roth (Karlsruhe, Germany) and Merck (Darmstadt, Germany), respectively. Ammonium formate was obtained from Fisher (Schwerte, Germany); ammonium acetate, D2O, DMSO-d6, sodium acetate and ammonia were from Sigma-Aldrich (St. Louis, MO, USA); ammonium hydroxide was from Grüssing (Filsum, Germany); and citric acid was from Carl Roth (Karlsruhe, Germany).
6
Proliferation Assay with L-Lys and L-Arg
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L-Lys (L-Lysine monohydrochloride) and L-Arg (L-Arginine) were purchased from Sigma-Aldrich (St. Louis, MO, USA), nor-NOHA from Bachem (Bubendorf, Switzerland). For proliferation assays, L-Arg-, L-Lys-phenol-free DMEM (Dulbecco's Modified Eagle's Medium; Sigma-Aldrich) was supplemented with 272 μM L-Lys.
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