2-Chlorotrityl chloride resin (reaction point density: 1.6 mmol g−1; Watanabe Chemical Industries, Hiroshima, Japan) was used as an insoluble resin carrier. 9-Fluorenylmethyloxycarbonyl-Phe (Fmoc-Phe, >99%, AAPPTEC), 9-fluorenylmethyloxycarbonyl-tyrosine (Fmoc-Tyr, >99%, AAPPTEC), N-Fmoc-N-trityl-L-histidine (Fmoc-His, >99%, Sigma-Aldrich, St. Louis, MA, USA), and 9-fluorenylmethyloxycarbonyl-glycine (Fmoc-Gly, AAPPTEC) were used as an extension of the peptide chain. 1-[Bis(dimethylamino)methyliumyl]-1H-1,2,3-triazolo [4,5-b]pyridine-3-oxide hexafluorophosphate (HATU, AAPPTEC) and diisopropylethylamine (DIEA, Fujifilm Wako Pure Chemicals, Osaka, Japan) were used as condensing agents. N,N-dimethylformamide (DMF), dichloromethane (DCM), methanol (MeOH), piperidine, trifluoroacetic acid (TFA), triisopropylsilane (TIS), and diethyl ether (Et2O), all purchased from Fujifilm Wako Pure Chemicals, were used for washing, cutting, and dissolving the Fmoc reagent. Ninhydrin and ethanol, also purchased from Fujifilm Wako Pure Chemicals, were used for the Ninhydrin color test.
Ninhydrin
Manufactured by Fujifilm
Sourced in Japan
Ninhydrin is a chemical reagent used in forensic science and analytical chemistry. It is primarily used to detect the presence of amino acids, which are the building blocks of proteins. Ninhydrin reacts with amino acids to produce a distinctive purple color, known as the 'Ruhemann's purple' reaction, which can be used to visualize and identify fingerprints, handwriting, and other trace evidence.
Automatically generated - may contain errors
4 protocols using ninhydrin
1
Solid-phase Peptide Synthesis Protocol
2
Amino Acid Analysis Using Hitachi L-8900
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Amino acid analysis was performed by the UC Davis Molecular Structure Facility using a Hitachi L-8900 amino acid analyzer (Hitachi High Tech, Tokyo, Japan). Samples were acidified to 2% sulfosalicylic acid (SSA) and incubated at room temperature (23 °C) for 15 min before being frozen (−20 °C) overnight. They were then diluted with 100 nmol/mL AE-Cys Li. 20 µL of sample was injected into the system, and free amino acids were separated using ion-exchange chromatography with a post-column ninhydrin reaction. Column and buffers were obtained from Hitachi, ninhydrin was supplied by Wako (Richmond, VA), and amino acid standards were obtained from Sigma-Aldrich (St. Louis, MO). Absorbance was recorded at both 570 nm and 440 nm after the reaction with ninhydrin to determine the response factor for each individual amino acid and to quantify levels relative to the known amino acid standards. AE-Cys was used to correct for injection volume variance. Certain amino acids such as tryptophan could not be measured using this method.
3
Quantitative Amino Acid Profiling
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Extracted sample pellets from JrGGT1- and JrGGT2-expressing and UT tobacco were dissolved in 100 nmol/mL AE-Cys (internal standard) Li (lithium) diluent before the 50-μL injection. Free amino acids were separated using strong cation-exchange chromatography with a post-column ninhydrin reaction for detection. Hitachi (Chiyoda, Tokyo, Japan) supplied column and buffers, and Wako (Richmond, Virginia) supplied ninhydrin. Calibration of the Hitachi 8900 was performed using amino acid standards (Sigma-Aldrich, St. Louis, MO, United States). Absorbance was recorded at both 570 nm and 440 nm after the reaction with ninhydrin to determine the response factor for each amino acid and to quantify relative to the known amino acid standards. The included internal standard (AE-Cys) was used to correct for any variance in injection volume due to the auto-sampler.
4
Plasma Amino Acid Analysis by HPLC
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Plasma nitrogen containing compounds were analyzed at Molecular Structure Facility, Proteomics Core of Genome Center (Davis, CA) with Li-based Hitachi 8900 according to established methods (Cooper et al., 2001 ). Briefly, plasma samples were thawed at 20 to 22 °C, acidified with 2% sulfosalicylic acid (Sigma #247006) final concentration and incubated at 20 to 22 °C for 15 min before overnight freezing the samples at −20 °C. Next day, the acidified samples were diluted with 100 nmol/mL aminoethylcysteine (AE-Cys) Li diluent (Pickering Labs, #Li220 Mountainview, CA) prior to the 50 μL injection. Free AA were separated using ion-exchange chromatography with a secondary post-column reaction with ninhydrin (WAKO, #299-70501). Column and buffers were supplied by Hitachi (Hitachi High-Technologies Corporation, Tokyo, Japan), and ninhydrin was supplied by Wako (FUJIFILM Wako Chemicals U.S.A. Corporation, Richmond, VA). Calibration of the Hitachi AA Analyzer (Model 8900, Japan) was performed using AA standards (Sigma–Aldrich, St. Louis, MO). Absorbance was recorded at both 570 and 440 nm after the reaction with ninhydrin to determine the response factor for each individual AA and to quantify levels relative to the known AA standards. The included internal standard (AE-Cys) was used to correct for any variations in injection volume caused by auto-sampler (integrated part of Hitachi 8900).
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