For TMT experiment, first 10 μg of each peptide sample was pooled for the reference peptide channel. Ten TMT 10plex reagents (Thermo Fisher) with different mass of reporter ions were labeled on N-terminus or amine group of Lysine amino acid, and the last channel of TMT-131 was used for reference peptides to normalize experimental batch effects. After quenching of excess TMT reagent using 5 % ammonium hydroxide (NH4OH), all channels in a batch were pooled in a tube followed by cleaning up salts using C18 column (Isolute-C18, Biotage). Eluted peptides were fractionated on HPLC (Ultimate 3000, Thermo Fisher) coupled with C18 column (ZORBAX 300Extend-C18, Agilent). Mobile phases were composed of 2% ACN, 15 mM NH4OH, pH 10 for Buffer A and 90% ACN, 15 mM NH4OH, pH 10 for Buffer B. Sample separation was accomplished using the following linear gradient: from 0% to 7% B in 2 min, from 7% to 45% B in 78 min, from 45% to 80% B in 5 min, and held at 80% B for an additional 10 min. Separated samples were collected in a 96 deep well plate and further concatenated into 24 fractions. The fractionated peptides were dried down and reconstituted in 0.1% FA with 3% ACN at a final concentration of 0.1 μg/μL for further analysis.
Isolute c18
Manufactured by Biotage
Sourced in Sweden, Italy
Isolute C18 is a reversed-phase solid-phase extraction (SPE) sorbent. It is designed for the extraction, purification, and cleanup of a wide range of organic compounds from various sample matrices.
Automatically generated - may contain errors
Lab products found in correlation
Quercetin 3 o arabinoside, by Biosynth (3 mentions)
Quercetin 3 o rhamnoside, by Biosynth (3 mentions)
Phloridzin, by Merck Group (2 mentions)
Quercetin 3 o galactoside, by Extrasynthese (2 mentions)
Chlorogenic acid, by Merck Group (2 mentions)
Hcx cartridges, by Biotage (2 mentions)
Tmt 10 plex reagent, by Thermo Fisher Scientific (1 mentions)
Ultimate 3000, by Thermo Fisher Scientific (1 mentions)
Zorbax 300extend c18, by Agilent Technologies (1 mentions)
Cyanidin chloride, by Merck Group (1 mentions)
Toluene, by Merck Group (1 mentions)
N methyl pyrrolidone (nmp), by Merck Group (1 mentions)
Piperidine, by Merck Group (1 mentions)
Piperazine, by Merck Group (1 mentions)
Oxyma, by Merck Group (1 mentions)
Fmoc gly oh, by Merck Group (1 mentions)
Fmoc trp oh, by Merck Group (1 mentions)
Fmoc pro oh, by Merck Group (1 mentions)
Fmoc trp boc oh, by Merck Group (1 mentions)
Fmoc tyr tbu oh, by Merck Group (1 mentions)
10 protocols using isolute c18
1
TMT-based Quantitative Proteomics
2
Extraction and Quantification of Phenolic Compounds
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Methanol (MeOH), formic acid (HCOOH), gallic acid, chlorogenic acid, cyanidin chloride and phloridzin were purchased from Sigma-Aldrich (Milan, Italy). Quercetin-3-O-galactoside was obtained from ExtraSynthese (Lyon, France). Quercetin-3-O-arabinoside, quercetin-3-O-xyloside and quercetin-3-O-rhamnoside were purchased from Carbosynth (Berkshire, UK). Milli-Q® grade water was produced by the Elgastat UHQ-PS system (ELGA, HighWycombe Bucks, UK). Solid phase extraction (SPE) columns ISOLUTE C18, 1 g, 6 mL were from Biotage (Milan, Italy).
3
Peptide Synthesis Protocol Compendium
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Fmoc-Gly-OH (≥98.0%), Fmoc-Gln(Trt)-OH
(≥98.0%), Fmoc-Trp-OH (≥97.0%), Fmoc-Trp(Boc)-OH (≥97.0%),
Fmoc-Pro-OH (≥99.0%), Fmoc-Tyr(tBu)-OH (≥98.0%),
DIC (99%), Oxyma (97%), NMP (≥99.0%), toluene (≥99.5%),
piperidine (99%), piperazine (99%), and DBU (98%) were purchased from
Sigma-Aldrich; 2-Cl-trityl chloride resin (100–200 mesh, 1%
DVB, 1.0–1.6 mmol/g), Fmoc-His(Trt)-OH (≥98.0%), and
Fmoc-Asn(Trt)-OH were procured from Iris Biotech GmbH; Fmoc-Cys[(CH2)3COOtBu]-OH (≥98.5%) was obtained from
Flamma, Dalian HonKai Chemical Development and Fmoc–N-4-F-Bn-Gly-OH (≥98.5%) was purchased from PolyPeptide
Group; Fmoc-Arg(Pbf)-OH (≥98.5%) was purchase from Flamma;
Fmoc-Sar-OH (≥98.0%) was procured from Fluorochem; DMF (≥99.5%)
was purchased from Merck; and Isolute C18 was procured from Biotage.
(≥98.0%), Fmoc-Trp-OH (≥97.0%), Fmoc-Trp(Boc)-OH (≥97.0%),
Fmoc-Pro-OH (≥99.0%), Fmoc-Tyr(tBu)-OH (≥98.0%),
DIC (99%), Oxyma (97%), NMP (≥99.0%), toluene (≥99.5%),
piperidine (99%), piperazine (99%), and DBU (98%) were purchased from
Sigma-Aldrich; 2-Cl-trityl chloride resin (100–200 mesh, 1%
DVB, 1.0–1.6 mmol/g), Fmoc-His(Trt)-OH (≥98.0%), and
Fmoc-Asn(Trt)-OH were procured from Iris Biotech GmbH; Fmoc-Cys[(CH2)3COOtBu]-OH (≥98.5%) was obtained from
Flamma, Dalian HonKai Chemical Development and Fmoc–N-4-F-Bn-Gly-OH (≥98.5%) was purchased from PolyPeptide
Group; Fmoc-Arg(Pbf)-OH (≥98.5%) was purchase from Flamma;
Fmoc-Sar-OH (≥98.0%) was procured from Fluorochem; DMF (≥99.5%)
was purchased from Merck; and Isolute C18 was procured from Biotage.
4
Solid Phase Extraction for Sample Cleanup
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The Clean–up of the extract was performed using a solid phase extraction (C18) column (ISOLUTE/C18 (100 mg/10 ml–1 ml XL), Biotage, Uppsala, Sweden). The column was conditioned using 1 ml of MeOH (10 %). A volume of 6 ml of the extract was added to the column using a vacuum manifold. Elution was performed twice using 1.5 ml of acetonitrile–methanol (40%) (optimized) and soaked for 2 min. The eluted sample was dried by evaporation and re–dissolved in 500 μL of the isocratic mobile phase (A:B, 1:1). Prior to loading the sample in LC-MS/MS, the samples were filtered through a syringe filter (0.45μm).
5
Oxylipins and Endocannabinoids Extraction
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Oxilipins and endocannabinoids were isolated from serum samples using an automated C18 solid phase extraction manifold (Biotage, Uppsala, Sweden). For this, 100 μL of patient samples was transferred to a deep well 96 well plate. To the sample, 300 μL of 20:80 methanol:water, 55 μL 1% BHT, and 80 μL of glacial acetic acid was added to adjust the pH to 3.0. Samples are centrifuged at 4000 rpm for 10 min at 4 °C to pellet any precipitated solids. The supernatant was transferred to a preconditioned C18 solid phase extraction (SPE) plate (Isolute C18, Biotage, Uppsala, Sweden). The sample was then rinsed with 800 μL water, followed by 800 μL hexane. Oxylipins and endocannabinoids were then eluted with 400 μL methyl formate, dried under nitrogen gas, and subsequently reconstituted in 200 μL methanol for LC/MS analysis.
6
Oxylipin Extraction from Plasma
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Oxilipins were isolated from plasma samples using an automated C18 solid phase extraction manifold, the Biotage Extrahera (Biotage, Uppsala, Sweden). Samples were prepared by pipetting 100 µL of patient plasma to a 96 well plate. To the sample, 300 µL of 20:80 methanol: water, 55 μL 1% butylated hydroxytoluene (BHT), and 80 µL of glacial acetic acid was added to achieve pH 3.0. Samples were centrifuged at 1750 × g for 10 minutes at 4 °C to pellet any precipitated solids. The supernatant was transferred to a C18 SPE plate (Isolute C18, Biotage, Uppsala, Sweden) that was preconditioned with 1 mL ethyl acetate and 1 mL of 95:5 water: methanol. After depositing the sample on the SPE plate, the sample was rinsed with 800 µL water, followed by 800 µL hexane. The oxylipins were then eluted off the SPE column with 400 µL methyl formate. The recovered oxylipin fraction was then dried under nitrogen gas and subsequently reconstituted in 100 μl methanol to be analyzed by LC/MS.
7
Enzymatic Characterization of Novel Psychoactive Substances
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Chemicals and reagents 6-APB, 5-APB, and 5-MAPB were synthesized [6] and provided by the Department of Pharmacology and Therapeutics, Trinity Centre for Health Sciences, St. James's Hospital (Dublin, Ireland), before 5-APB and 6-APB were scheduled. 6-MAPB was synthesized and provided by the School of Pharmacy and Biomolecular Sciences, Liverpool John Moores University (Liverpool, UK). Isolute C18 (500 mg, 3 mL) and HCX cartridges (130 mg, 3 mL) were obtained from Biotage (Uppsala, Sweden), isocitrate and isocitrate dehydrogenase, heptafluorobutyric anhydride (HFBA), sodium phosphate from Sigma (Taufkirchen, Germany), NADP + from Biomol (Hamburg, Germany), acetonitrile (LC-MS grade), ammonium formate (analytical grade), formic acid (LC-MS grade), methanol (LC-MS grade), mixture (100,000 Fishman units/mL) of glucuronidase (EC No. from VWR (Darmstadt, Germany). The baculovirus-infected insect cell microsomes (Supersomes) containing 1 nmol/mL of human cDNA-expressed CYP 1A2, CYP 2A6, CYP 2B6, CYP 2C8, CYP 2C9, CYP 2C19, CYP 2D6, CYP 2E1 (2 nmol/mL), CYP 3A4, or CYP 3A5 (2 nmol/mL), and pooled human liver microsomes (pHLM, 20 mg microsomal protein/mL, 400 pmol total CYP/mg protein) were obtained from BD Biosciences (Heidelberg, Germany). After delivery, the microsomes were thawed at 37°C, aliquoted, snapfrozen in liquid nitrogen, and stored at -80°C until use.
8
Phytochemical Profiling and Bioactivity
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Methanol (MeOH), formic acid (HCOOH), acetic acid (CH3COOH), fructose, glucose, sucrose, (+)-catechin, (-)epicatechin, chlorogenic acid, phloridzin, phloretin, 3hydroxycinnamic acid (internal standard; I.S.), α-glucosidase, . Quercetin-3-O-arabinoside and quercetin-3-Orhamnoside were purchased from Carbosynth (Berkshire, UK). Milli-Q grade water was produced by Elgastat UHQ-PS system (ELGA, High Wycombe Bucks, UK). Solid phase extraction (SPE) columns ISOLUTE C18, 1 g, 6 mL were from Biotage (Milan, Italy). 10 kDa cut off sacks (Avg. flat width 35 mm) were purchased from Sigma-Aldrich (Milan, Italy). Human serum albumin (>99%) was obtained from Sigma.
9
Phytochemical Analysis of Plant Extracts
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Methanol (MeOH), formic acid (HCOOH), fructose, glucose, sucrose, (+)-catechin, (-)-epicatechin, chlorogenic acid, phloridzin, phloretin, 3-hydroxycinnamic acid (internal standard; I.S.), glucosidase, 4-nitrophenyl--D-glucopyranoside, and acarbose were purchased from Sigma-Aldrich (Milan, Italy). Quercetin-3-O-galactoside, procyanidin B2, and epigallocatechin gallate were obtained from ExtraSynthese (Lyon, France). Quercetin-3-O-arabinoside and quercetin-3-Orhamnoside were purchased from Carbosynth (Berkshire, UK). Milli-Q grade water was produced by Elgastat UHQ-PS system (ELGA, High Wycombe Bucks, UK).
Solid phase extraction (SPE) columns ISOLUTE C18, 1 g, 6 mL were from Biotage (Milan, Italy).
Solid phase extraction (SPE) columns ISOLUTE C18, 1 g, 6 mL were from Biotage (Milan, Italy).
10
Comprehensive LC-MS/MS Analysis of Illicit Drugs in Environmental Urine
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Chemicals and reagents COC, COC-d 3 , cocaethylene (CE), CE-d 3 , BE, BE-d 3 , MDEA-d 5 , and HMMA were obtained by LGC standards, ecgonine methyl ester (EME) and EME-d 3 were obtained from Cerilliant (Round Rock, TX, USA). HMMA-d 3 , MDMA, MDMA-d 5 , 3,4methylenedioxymethamphetamine (MDA), and MDA-d 5 were obtained from Sigma-Aldrich (St Louis, MO, USA). HMMA sulfate (HMMA-S) was produced as described previously. [17] Hydroxymethoxyethylamphetamine (HMEA) was produced as described previously. [18] MDEA was obtained from seized materials. Dilutions and storage of COC and its metabolites were done in acetonitrile, for MDMA and metabolites in methanol. Isolute C18 (500 mg, 3 mL) and HCX cartridges (130 mg, 3 mL) were obtained from Biotage (Uppsala, Sweden). Mixture (100 All PU samples were spiked on site with HMMA-d 3 to a final concentration of 100 ng/mL and stored in polystyrene containers and immediately put on ice until freezing (max 12 h).
US was collected from areas (fences, bushes, etc.) where pools of urine were visible on the surface. The characteristics (liquid content, colour, type of soil) differed from urine with small amounts of sand to humus with high organic content.
US was collected from areas (fences, bushes, etc.) where pools of urine were visible on the surface. The characteristics (liquid content, colour, type of soil) differed from urine with small amounts of sand to humus with high organic content.
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