Cysgly
Manufactured by Merck Group
Sourced in United States
CysGly is a lab equipment product manufactured by Merck Group. It is a dipeptide composed of the amino acids cysteine and glycine. CysGly serves as a substrate for enzymatic reactions and is used in various biochemical and analytical applications.
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Lab products found in correlation
L gsh, by Merck Group (3 mentions)
γ glucys, by Merck Group (3 mentions)
Glutathione (gsh), by Merck Group (3 mentions)
Milli q system, by Merck Group (2 mentions)
Phosphate buffered saline (pbs), by Takara Bio (2 mentions)
Ammonium 7 fluoro 2 1 3 benzoxadiazole 4 sulfonate sbd f, by Dojindo Laboratories (2 mentions)
L cys, by Merck Group (2 mentions)
Dl hcy, by Merck Group (2 mentions)
Trichloroacetic acid, by Fujifilm (1 mentions)
N acetyl l cysteine, by Fujifilm (1 mentions)
Anhydrous pyridine, by Merck Group (1 mentions)
Sodium chloride (nacl), by Merck Group (1 mentions)
Dithiothreitol (dtt), by Merck Group (1 mentions)
Human serum albumin (hsa), by Merck Group (1 mentions)
Mstfa, by Merck Group (1 mentions)
Lc 30ad, by Shimadzu (1 mentions)
Lcms 2020, by Shimadzu (1 mentions)
Inertsustain, by GL Sciences (1 mentions)
5 oxoproline, by Merck Group (1 mentions)
Lc ms grade water, by Honeywell (1 mentions)
10 protocols using cysgly
1
Quantification of Thiol Compounds
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l -Cys, D ,l -Hcy, l -GSH, CysGly, and γGluCys were purchased from Sigma-Aldrich (St. Louis, MO, USA). N-Acetyl-l-Cysteine (NAC), tiopronin (N-(2-mercaptopropionyl)glycine, MPG), and trichloroacetic acid (TCA) were obtained from Wako (Osaka, Japan). Ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate (SBD-F) was purchased from Dojindo (Kumamoto, Japan). TCEP was from Tokyo Chemical Industry (Tokyo, Japan). Phosphate buffered saline (PBS) was purchased from Takara Bio (Shiga, Japan). HPLC-grade acetonitrile was used. Water was purified using a Milli-Q system (Millipore, Bedford, MA, USA). All other chemicals were of analytical grade.
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2
Analysis of Thiol-Based Compounds
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All chemicals used throughout this study were of analytical reagent grade. D,L-HTL, D,L-Met, CysGly, symmetrical disulfides of D,L-Hcy, D,L-Cys, and L-GSH, MSTFA, TMCS, TCEP, DTT, 2-ME, THP, HSA, sodium chloride, and anhydrous pyridine were from Sigma-Aldrich, (St. Louis, MO, USA). PCA, hydrochloric acid, acetic acid, sodium hydroxide, HPLC-gradient grade MeCN, ethanol, chloroform, methanol, sodium hydrogen phosphate heptahydrate, sodium dihydrogen phosphate dihydrate were from J.T. (Baker, Deventer), the Netherlands. CMLT was prepared as previously described [24 (link)]. Deionized water was produced in our laboratory.
3
Identifying GSH Degradation by LC-MS
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To identify degradation products of GSH by using LC-MS, the products were confirmed by the selected-ion-monitoring (SIM) mode. The m/z values of [M+H]+ were adopted for the LC-MS-SIM. The LC-MS system consisted of a high-performance liquid chromatography (HPLC) device, LC-30AD (Shimadzu, Kyoto, Japan), and an MS detector with an electrospray ion source and quadrupole analyzer, LCMS-2020 (Shimadzu). LC-MS was carried out using an InertSustain column (4.6 by 50 mm; GL Sciences, Tokyo, Japan). The elution was performed with 0.1% formic acid for 5 min; the flow rate, column temperature, and injection volume were 0.5 ml/min, 35°C, and 5 µl, respectively. All samples were injected by 5× dilution with 0.1% formic acid. GSH (1 mM) (Sigma), 5-oxoproline (Sigma), and Cys-Gly (Sigma) were used as authentic standards.
4
Glutathione and Cysteine Derivatives Analysis
5
Genetic Manipulation of Human GGT1
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The expression plasmid pCXN2 vector was a generous gift from Dr J. Miyazaki (Osaka University). The coding sequence of human GGT1 was amplified from HeLa cells by reverse transcription–PCR and subcloned into pCXN2. The single-guide RNAs (sgRNAs) targeting human GGT1 sequence (#1, 5′-actgcgggacggtggctctg-3’; #2, 5′-ctgcttggcatccgcggcca-3′) were cloned into the sgRNA expression vector peSpCAS9(1.1)-2xsgRNA (Addgene; plasmid no.: 80768). Cystine-free medium was prepared as described previously (15 (link)). Inhibitors and amino acid compounds were used at the following concentrations: BSO, 100 μM; DFO, 100 μM (Santa Cruz Biotechnology); Fer-1, 1 μM; SSZ, 250 μM; Cys-Gly, 100 μM; glutamate, 100 μM (Sigma–Aldrich); OU749, 250 μM; NCT-503, 25 μM (Cayman); and GSH (Nacalai Tesque, Inc), 100 μM.
6
Quantification of Thiol-Containing Compounds
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Cystamine dihydrochloride, cysteamine (≥ 98%), Cys (≥ 98%), CysGly (≥ 85%), reduced GSH (≥ 98%), HCys (≥ 95%), acetic acid, sodium acetate, boric acid, trichloroacetic acid, EDTA and phosphate buffered saline were purchased from Sigma-Aldrich (Dorset, UK). Sodium hydroxide (50% concentrated solution) and 7-fluorobenzofurazan-4-sulfonic acid were from Fluka (Sigma-Aldrich). Analytical reagent-grade hydrochloric acid and HPLC-grade methanol were from Fisher Scientific (Loughborough, UK) and tris(2-carboxyethyl)phosphine hydrochloride was from Thermo Fisher Scientific (Waltham, MA, USA). Milli-Q water (18.2 MΩ cm−1) was used in all experiments (Merck Millipore UK, Watford, UK). Sulfate conjugates were synthesized as previously described53 (link). FA-gly and VA-gly were chemically synthesized and fully characterised, and this will be published elsewhere.
7
Quantification of Sulforaphane Metabolites
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LC–MS-grade acetonitrile was from Burdick and Jackson (Muskegon, MI, USA). LC–MS-grade formic acid (OptimaTM) was from Fisher Chemical (Thermo Fisher Scientific, Waltham, MA, USA). Reverse osmosis purified MilliQ water used in LC–MS analysis was from Millipore water purification system (Merck, Darmstadt, Germany). Analytical standards SFN, SFN-d8, SFN-NAC-d8, SFN-GSH, SFN-NAC, SFN-Cys were from Toronto Research Chemicals (Toronto Research Chemicals, Toronto, ON, Canada). CysGly (>85%) and pyridine were purchased from Sigma Aldrich (St. Louis, MI, USA). HF Bond EluteTM SPE cartridges C18 (6 mL tube, 500 mg bed) were purchased from Agilent Technologies (Colorado Springs, CO, USA). Myrosinase-activated broccoli sprout extract capsules, BroccomaxTM, were sourced from Jarrow Formulas (Los Angeles, CA, USA).
8
Quantification of Thiol-Containing Compounds
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L -Cys, D ,L -Hcy, L -GSH, and CysGly were purchased from Sigma-Aldrich (St. Louis, MO, USA). Trichloroacetic acid (TCA) was obtained from Wako Pure Chemical (Osaka, Japan). Ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate (SBD-F) was purchased from Dojindo (Kumamoto, Japan). TCEP was purchased from the Tokyo Chemical Industry (Tokyo, Japan). Phosphate buffered saline (PBS) was purchased from Takara Bio (Shiga, Japan). HPLC-grade acetonitrile was used. Water was purified using a Milli-Q system (Millipore, Bedford, MA, USA). All other chemicals were of analytical grade.
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9
Quantification of Deoxynivalenol Metabolites
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DON (≥98%), GSH (≥98%), Cys, γ-GluCys, CysGly, NAC and Na2CO3 (pro analysis) were from Sigma-Aldrich (Steinheim, Germany), and NaHCO3 (pro analysis) was from Fluka (Steinheim, Germany). NaHCO3 and Na2CO3 were used to prepare 0.2 M buffer with pH of 10.7 as measured with a Mettler Delta 320 pH meter at ambient temperature. Purified quantitative standards of DON-10-Cys, DON-10-GSH, DON-13-Cys and DON-13-GSH were available from previous work [6 (link),7 (link)], while DON-3-O-β-d -glucoside, DON-3-O-acetate and DON-15-O-acetate were from Romer Labs (Tulln, Austria).
10
Oxidative Stress Enzyme Assays
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GSH, Cys, CysGly, ȖGluCys, BSA, SDS, 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), trans-2nonenal, NADP + , NADPH, 9,10-PQ, and sequencing-grade trypsinwere purchased from Sigma Aldrich (St. Louis, MO, USA). 4-Oxo-nonenal (ONE), prostaglandin B 1 (PGB 1 ) and 4-hydroxy-2nonenal mercapturic acid were purchased from Cayman Chemicals (Ann Arbor, MI, USA) Whatman DEAE-cellulose (DE-52) and Sephacryl S200 were purchased from GE Healthcare (Little Chalfont, UK). Blue Sepharose and Bradford reagent were purchased from Bio-Rad (Hercules, CA, USA). YM10 membranes (10kDa cut-off) were purchased from Amicon Millipore (Darmstadt, Germany). RPMI 1640, penicillin, streptomycin, glutamine and foetal bovine serum (FBS) were obtained from Lonza (Basel, Switzerland). Dialysis tubing (10kDa cut-off) was purchased from Spectrum Laboratories Inc (Rancho Dominguez, CA, USA). All inorganic chemicals were of reagent grade from BDH (VWR international ltd, Poole Dorset, UK). All solvents were HPLCgrade from J.T. Baker Chemicals (USA).
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