Extraction of PCB 136 and its hydroxylated metabolites was performed using a published method.36 (link), 37 (link) In short, surrogate standards (2,3,4,4',5,6-hexachlorobiphenyl, 500 ng; 2',3,3',4,5,5'-hexachlorobiphenyl-4-ol, 274 ng) were added to each sample, followed by hydrochloric acid (6 M, 1 mL) and 2-propanol (3 mL). The samples were extracted with hexane-MTBE (1:1 v/v, 5 mL) and hexane (3 mL), and the combined organic extracts were washed with an aqueous KCl solution (1%, 3 mL). After removal of the organic phase, the KCl phase was re-extracted with hexane (3 mL), and the combined extracts were reduced under a gentle stream of nitrogen to ~ 1 mL. After addition of methanol (5 drops), the hydroxylated PCBs were derivatized with diazomethane in diethyl ether (0.5 mL) and subjected to a sulfur cleanup as described previously.36 (link) The mean recovery rates were 93 ± 17 % and 93 ± 16 % for 2,3,4,4',5,6-hexachlorobiphenyl and 2',3,3',4,5,5'-hexachlorobiphenyl-4-ol, respectively. The concentrations were corrected for recovery rates below 100 %. Concentrations of PCB 136 and its metabolites were determined using PCB 204 as internal standard. All data are presented as mean ± SD (n = 3).
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Organic Chemical
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Diazomethane
Diazomethane
Diazomethane is a highly reactive organic compound used in various chemical reactions, particularly in the synthesis of organic compounds.
It is a powerful methylating agent and is commonly employed in the preparation of esters, ethers, and other derivatives.
Diazomethane has a wide range of applications in organic chemistry research and is an important tool for chemists investigating complex molecular structures.
Due to its reactive nature, proper safety precautions are essential when handling diazomethane.
Researchers can now leverage PubCompare.ai to optimize thier diazomethane research, easily locating relevant protocols from literature, preprints, and patents, and using AI-driven comparisons to identify the most accurate and reproducible methods.
This powerful tool can help enhance the accuracy and reproducibility of diazomethane-based studies.
It is a powerful methylating agent and is commonly employed in the preparation of esters, ethers, and other derivatives.
Diazomethane has a wide range of applications in organic chemistry research and is an important tool for chemists investigating complex molecular structures.
Due to its reactive nature, proper safety precautions are essential when handling diazomethane.
Researchers can now leverage PubCompare.ai to optimize thier diazomethane research, easily locating relevant protocols from literature, preprints, and patents, and using AI-driven comparisons to identify the most accurate and reproducible methods.
This powerful tool can help enhance the accuracy and reproducibility of diazomethane-based studies.
Most cited protocols related to «Diazomethane»
Diazomethane
Ethyl Ether
Hydrochloric acid
Isopropyl Alcohol
Methanol
methyl tert-butyl ether
n-hexane
Nitrogen
PCB 136
Sulfur
750,000 Mcf10a cells grown on a 35 mm dish were killed in 750 μL ice-cold 1 M HCL, then scraped and collected into an Eppendorf tube. Each sample was then split into three separate 2 mL polypropylene Eppendorf tubes; 250 μL for PI, PIP, PIP2, PIP3 measurement, 250 μL for PI(3,4)P2/PI(4,5)P2 measurement, and the remaining cells were kept for analysis by western blot. Cells were pelleted in a microfuge (15,000 x g, 10 min at 4°C), the supernatant removed and cell pellets either processed immediately or snap-frozen in liquid nitrogen and stored at −80°C for up to two weeks.
For human prostate and breast cancer cell lines, 250,000 cells were seeded into 35 mm dishes and grown in the medium optimal for each cell line for 32h. Cells were then starved for 16h by replacing the medium with starvation medium – a phenol red-free RPMI 1640 supplemented with 2mM glutamax. Following stimulation and / or inhibition with appropriate reagents, medium was removed by aspiration and cells killed with 750 μl ice-cold 1M HCL. Cells were then scraped, collected into Eppendorf tubes, pelleted, and snap-frozen, as described above.
920 μL of a solvent mixture containing 2:1:0.79 (v/v) MeOH:CHCl3:H2O(acidic) was added to the cell pellets and the mixture vortexed thoroughly for 10 s. Relevant internal standards were then added:10 ng C17:0/C16:0-PIP3, 100 ng C17:0/C16:0-PI, 250 ng d6-C18:0/C20:4-PI(4,5)P2 for routine analysis of PI, PIP, PIP2 and PIP3; 50 ng C17:0/C20:4 PI, 50 ng d6- C18:0/C20:4-PI(3,4)P2, 250 ng d6-C18:0/C20:4-PI(4,5)P2 for routine analysis of PI, PI(3,4)P2 and PI(4,5)P2. Lipids were then extracted using an acidified Folch phase partition and derivatised with TMS-diazomethane (Clark et al., 2011 (link)).
Molecules derived from PI, PIP, PIP2, and PIP3 were measured by HPLC-MS (Kielkowska et al., 2014 (link)). Response ratios were calculated for the endogenous species of these lipids divided by their relevant C17:0/C16:0 internal standard. We routinely analyzed 5 molecular species of these lipids but present here data for the C38:4 species only, to align with data presented for the C38:4 species of PI(3,4)P2 and PI(4,5)P2 (see below). The C38:4 species of PIP2 and PIP3 represent approx. 10%–15% of the total species of these lipids in Mcf10a cells and all species behave very similarly upon stimulation with EGF (Anderson et al., 2016 (link)). In some experiments, absolute amounts of C38:4 PI(3,4,5)P3 were generated by reference to standard curves previously generated for this molecular species (Kielkowska et al., 2014 (link)). Three technical replicates were routinely analyzed for each experiment and, unless stated otherwise, data are presented as means SD of three biological replicates.
Molecules derived from PI, PI(3,4)P2 and PI(4,5)P2 were analyzed by a new HPLC-MS method, see below. Response ratios were calculated for the endogenous C38:4 species of these lipids divided by their relevant d6-labeled internal standard. In some experiments, absolute amounts of these lipids were generated by reference to standard curves (Figure S1 ). Three technical replicates were routinely analyzed for each experiment and, unless stated otherwise, data are presented as means SD of three biological replicates.
For human prostate and breast cancer cell lines, 250,000 cells were seeded into 35 mm dishes and grown in the medium optimal for each cell line for 32h. Cells were then starved for 16h by replacing the medium with starvation medium – a phenol red-free RPMI 1640 supplemented with 2mM glutamax. Following stimulation and / or inhibition with appropriate reagents, medium was removed by aspiration and cells killed with 750 μl ice-cold 1M HCL. Cells were then scraped, collected into Eppendorf tubes, pelleted, and snap-frozen, as described above.
920 μL of a solvent mixture containing 2:1:0.79 (v/v) MeOH:CHCl3:H2O(acidic) was added to the cell pellets and the mixture vortexed thoroughly for 10 s. Relevant internal standards were then added:10 ng C17:0/C16:0-PIP3, 100 ng C17:0/C16:0-PI, 250 ng d6-C18:0/C20:4-PI(4,5)P2 for routine analysis of PI, PIP, PIP2 and PIP3; 50 ng C17:0/C20:4 PI, 50 ng d6- C18:0/C20:4-PI(3,4)P2, 250 ng d6-C18:0/C20:4-PI(4,5)P2 for routine analysis of PI, PI(3,4)P2 and PI(4,5)P2. Lipids were then extracted using an acidified Folch phase partition and derivatised with TMS-diazomethane (Clark et al., 2011 (link)).
Molecules derived from PI, PIP, PIP2, and PIP3 were measured by HPLC-MS (Kielkowska et al., 2014 (link)). Response ratios were calculated for the endogenous species of these lipids divided by their relevant C17:0/C16:0 internal standard. We routinely analyzed 5 molecular species of these lipids but present here data for the C38:4 species only, to align with data presented for the C38:4 species of PI(3,4)P2 and PI(4,5)P2 (see below). The C38:4 species of PIP2 and PIP3 represent approx. 10%–15% of the total species of these lipids in Mcf10a cells and all species behave very similarly upon stimulation with EGF (Anderson et al., 2016 (link)). In some experiments, absolute amounts of C38:4 PI(3,4,5)P3 were generated by reference to standard curves previously generated for this molecular species (Kielkowska et al., 2014 (link)). Three technical replicates were routinely analyzed for each experiment and, unless stated otherwise, data are presented as means SD of three biological replicates.
Molecules derived from PI, PI(3,4)P2 and PI(4,5)P2 were analyzed by a new HPLC-MS method, see below. Response ratios were calculated for the endogenous C38:4 species of these lipids divided by their relevant d6-labeled internal standard. In some experiments, absolute amounts of these lipids were generated by reference to standard curves (
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Acids
Biopharmaceuticals
Cell Lines
Cells
Chloroform
Cold Temperature
Diazomethane
Freezing
High-Performance Liquid Chromatographies
Homo sapiens
Hyperostosis, Diffuse Idiopathic Skeletal
Lipids
MCF-7 Cells
Nitrogen
Pellets, Drug
Polypropylenes
Prostate
Psychological Inhibition
Solvents
Western Blot
To quantify plant endogenous ABA and SA content, the ABA and SA of inoculated and non-inoculated samples were extracted from freeze-dried samples (three replicates). The quantification and extraction of ABA were performed using a previous protocol [63 (link),64 (link)] with some modifications. Around 150 mg of powder was homogenized in 2 mL of 90% methanol including 15 mg of butylated hydroxytoluene and 20 mL of 2% glacial acetic acid. The homogenate was incubated for 48 h at 4 °C, dried using a rotary evaporator, and methylated with diazomethane for further analysis. ABA was quantitatively assessed by GC-MS/SIM (5973 Network Mass Selective Detector and 6890N Network GC System; Agilent Technologies, Palo Alto, CA, USA) in three identical repeats.
The lyophilized sample was further crushed into fine powder in liquid nitrogen for SA quantification following a previous method [65 (link)]. Additionally, the powdered sample (0.2 g) was mixed with 2 mL of 90% methanol (Sigma, Germany) and centrifuged for 20 min at 10,000× g. The methanol in the supernatant was evaporated in a vacuum centrifuge and the sample was resuspended in 3 mL of 5% trichloroacetic acid (Sigma, Germany). The upper organic layer was further mixed with a solution of isopropanol, ethyl acetate, and cyclopentane (1:49.5:49.5 v/v) (Duksan, South Korea) and vigorously vortexed. The uppermost layer was transferred to a 4 mL tube and vacuum dried. Prior to high-performance liquid chromatography (HPLC), the dried pellet was mixed with 1 mL of HPLC mobile phase and SA was quantified through fluorescence detection.
The lyophilized sample was further crushed into fine powder in liquid nitrogen for SA quantification following a previous method [65 (link)]. Additionally, the powdered sample (0.2 g) was mixed with 2 mL of 90% methanol (Sigma, Germany) and centrifuged for 20 min at 10,000× g. The methanol in the supernatant was evaporated in a vacuum centrifuge and the sample was resuspended in 3 mL of 5% trichloroacetic acid (Sigma, Germany). The upper organic layer was further mixed with a solution of isopropanol, ethyl acetate, and cyclopentane (1:49.5:49.5 v/v) (Duksan, South Korea) and vigorously vortexed. The uppermost layer was transferred to a 4 mL tube and vacuum dried. Prior to high-performance liquid chromatography (HPLC), the dried pellet was mixed with 1 mL of HPLC mobile phase and SA was quantified through fluorescence detection.
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Acetic Acid
Cyclopentane
Diazomethane
ethyl acetate
Fluorescence
Freezing
Gas Chromatography-Mass Spectrometry
High-Performance Liquid Chromatographies
Hydroxytoluene, Butylated
Isopropyl Alcohol
Methanol
Nitrogen
Plants
Trichloroacetic Acid
Vacuum
Serum samples and survey data were collected from junior high school-aged
students and their mothers who were enrolled in the Airborne Exposures
to Semivolatile Organic Pollutants (AESOP) Study between April 2009
and March 2010. In this second year of the study, serum was analyzed
from 50 East Chicago mothers and their 50 enrolled children and from
46 Columbus Junction area mothers and their 54 enrolled children.
Of those 200 participants, 155 had also provided blood for the year
1 (April 2008-March 2009) data set. Nine families enrolled more than
one child. All AESOP subjects gave informed consent or assent in English
or Spanish according to an established Institutional Review Board
protocol. Participants generally did not fast prior to giving blood.
The sample collection, extraction, separation, and cleanup methods
are described in detail elsewhere,2 (link) with
minor improvements included here. Briefly, sera were weighed (∼4
g) and spiked with 5 ng 13C-labeled PCBs and 4′-OH-PCB
159 (Supporting Information (SI) Table
S1). The OH-PCB extract was derivatized to the methoxylated form (MeO-PCBs)
using diazomethane. Immediately prior to instrument analysis, PCB
extracts were spiked with 2 ng 13C-labeled internal standards
and OH-PCB extracts were spiked with 5 ng PCB 209 (SI Table S1). Nine samples were removed from the PCB and OH-PCB
data sets for having less than 4 g serum available for extraction,
and 33 samples were removed from the OH-PCB data set following extraction
errors.
GC-MS/MS (Agilent 7000 and Agilent 6890N with Waters
Micromass MS) in multiple reaction monitoring mode was used for identification
and quantification of 209 PCB congeners as 159 chromatographic peaks.
GC-ECD was used for identification and quantification of 12 OH-PCB
congeners as MeO-PCBs. Instrument operating parameters are in theSI . Instrument blanks of hexane were analyzed
with each instrument run before and after the calibration and after
the samples to ensure no cross-contamination.
Calibration standards
were purchased from Cambridge Isotope Laboratories,
Inc. (Andover, MA) and AccuStandard, Inc. (New Haven, CT, USA). The
OH-PCB congeners were chosen based on the known metabolic pathways
for the most common PCB congeners detected in the year 1 serum samples
and commercial availability (as MeO-PCBs). Congener mass was calculated
by applying a relative response factor obtained from each congener
in the calibration.
A common congener list (SI and Table
S7) was used when comparing the two data sets. Median change in PCB
concentration from year 1 to year 2 was 8 ng/g lw (28%) considering
all congeners and 6 ng/g lw (14%) using the common congener list.
Median change in OH-PCB concentration from year 1 to year 2 was 0.032
ng/g fw (54%) considering all congeners and 0.004 ng/g fw (4%) using
the common congener list.
students and their mothers who were enrolled in the Airborne Exposures
to Semivolatile Organic Pollutants (AESOP) Study between April 2009
and March 2010. In this second year of the study, serum was analyzed
from 50 East Chicago mothers and their 50 enrolled children and from
46 Columbus Junction area mothers and their 54 enrolled children.
Of those 200 participants, 155 had also provided blood for the year
1 (April 2008-March 2009) data set. Nine families enrolled more than
one child. All AESOP subjects gave informed consent or assent in English
or Spanish according to an established Institutional Review Board
protocol. Participants generally did not fast prior to giving blood.
The sample collection, extraction, separation, and cleanup methods
are described in detail elsewhere,2 (link) with
minor improvements included here. Briefly, sera were weighed (∼4
g) and spiked with 5 ng 13C-labeled PCBs and 4′-OH-PCB
159 (
S1). The OH-PCB extract was derivatized to the methoxylated form (MeO-PCBs)
using diazomethane. Immediately prior to instrument analysis, PCB
extracts were spiked with 2 ng 13C-labeled internal standards
and OH-PCB extracts were spiked with 5 ng PCB 209 (
data sets for having less than 4 g serum available for extraction,
and 33 samples were removed from the OH-PCB data set following extraction
errors.
GC-MS/MS (Agilent 7000 and Agilent 6890N with Waters
Micromass MS) in multiple reaction monitoring mode was used for identification
and quantification of 209 PCB congeners as 159 chromatographic peaks.
GC-ECD was used for identification and quantification of 12 OH-PCB
congeners as MeO-PCBs. Instrument operating parameters are in the
with each instrument run before and after the calibration and after
the samples to ensure no cross-contamination.
Calibration standards
were purchased from Cambridge Isotope Laboratories,
Inc. (Andover, MA) and AccuStandard, Inc. (New Haven, CT, USA). The
OH-PCB congeners were chosen based on the known metabolic pathways
for the most common PCB congeners detected in the year 1 serum samples
and commercial availability (as MeO-PCBs). Congener mass was calculated
by applying a relative response factor obtained from each congener
in the calibration.
A common congener list (
S7) was used when comparing the two data sets. Median change in PCB
concentration from year 1 to year 2 was 8 ng/g lw (28%) considering
all congeners and 6 ng/g lw (14%) using the common congener list.
Median change in OH-PCB concentration from year 1 to year 2 was 0.032
ng/g fw (54%) considering all congeners and 0.004 ng/g fw (4%) using
the common congener list.
BLOOD
Child
Chromatography
Diazomethane
Environmental Pollutants
Gas Chromatography-Mass Spectrometry
Hexanes
Hispanic or Latino
Isotopes
Mothers
Serum
Specimen Collection
The endogenous ABA content was quantified from the frozen samples by following the protocols of Qi et al.
[66 (link)] and Kamboj et al.
[67 ]. Aerial parts of the plant samples were extracted with 30 ml of extraction solution containing 95% isopropanol, 5% glacial acetic acid, and 20 ng of [(±)–3,5,5,7,7,7–d6]–ABA. The filtrate was concentrated by a rotary evaporator. The residue was dissolved in 4 ml of 1 N sodium hydroxide solution, and then washed three times with 3 ml of methylene chloride to remove lipophilic materials. The aqueous phase, brought to approximately a pH of 3.5 with 6 N hydrochloric acid was partitioned three times into ethyl acetate (EtOAc). EtOAc extracts were then combined and evaporated. The dried residue was dissolved in phosphate buffer (pH 8.0) and then run through a polyvinylpolypyrrolidone (PVPP) column. The phosphate buffer was adjusted to pH 3.5 with 6 N HCl and partitioned three times into EtOAc. EtOAc extracts were combined again and evaporated. The residue was dissolved in dichloromethane (CH2Cl2), and passed through a silica cartridge (Sep-Pak; Water Associates, Milford, Massachusetts, USA) pre-washed with 10 ml of diethyl ether: methanol (3:2, v/v) and 10 ml of dichloromethane. ABA was recovered from the cartridge by elution with 10 ml of diethyl ether (CH3-CH2)2O: methanol (MeOH) (3:2, v/v). The extracts were dried and methylated by adding diazomethane for GC/MS-SIM (6890 N network GC system, and the 5973 network mass-selective detector; Agilent Technologies, Palo Alto, CA, USA) analysis. For quantification, the Lab-Base (ThermoQuset, Manchester, UK) data system software was used to monitor responses to ions with an m/e of 162 and 190 for Me-ABA and 166 and 194 for Me-[2H6]-ABA.
[66 (link)] and Kamboj et al.
[67 ]. Aerial parts of the plant samples were extracted with 30 ml of extraction solution containing 95% isopropanol, 5% glacial acetic acid, and 20 ng of [(±)–3,5,5,7,7,7–d6]–ABA. The filtrate was concentrated by a rotary evaporator. The residue was dissolved in 4 ml of 1 N sodium hydroxide solution, and then washed three times with 3 ml of methylene chloride to remove lipophilic materials. The aqueous phase, brought to approximately a pH of 3.5 with 6 N hydrochloric acid was partitioned three times into ethyl acetate (EtOAc). EtOAc extracts were then combined and evaporated. The dried residue was dissolved in phosphate buffer (pH 8.0) and then run through a polyvinylpolypyrrolidone (PVPP) column. The phosphate buffer was adjusted to pH 3.5 with 6 N HCl and partitioned three times into EtOAc. EtOAc extracts were combined again and evaporated. The residue was dissolved in dichloromethane (CH2Cl2), and passed through a silica cartridge (Sep-Pak; Water Associates, Milford, Massachusetts, USA) pre-washed with 10 ml of diethyl ether: methanol (3:2, v/v) and 10 ml of dichloromethane. ABA was recovered from the cartridge by elution with 10 ml of diethyl ether (CH3-CH2)2O: methanol (MeOH) (3:2, v/v). The extracts were dried and methylated by adding diazomethane for GC/MS-SIM (6890 N network GC system, and the 5973 network mass-selective detector; Agilent Technologies, Palo Alto, CA, USA) analysis. For quantification, the Lab-Base (ThermoQuset, Manchester, UK) data system software was used to monitor responses to ions with an m/e of 162 and 190 for Me-ABA and 166 and 194 for Me-[2H6]-ABA.
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Acetic Acid
Buffers
Diazomethane
ethyl acetate
Ethyl Ether
Freezing
Gas Chromatography-Mass Spectrometry
Hydrochloric acid
Ions
Isopropyl Alcohol
Methanol
Methylene Chloride
Phosphates
Plant Components, Aerial
polyvinylpolypyrrolidone
Silicon Dioxide
Sodium Hydroxide
Most recents protocols related to «Diazomethane»
Example 1
1-palmitoyl-2-(dipyrrometheneboron difluoride) undecanoyl-sn-glycero-3-phosphoethanolamine (0.28 micromoles) and NHS-diazirine succinimidyl 4,4′-azipentanoate (2.8 micromoles) were mixed in chloroform (0.1 ml) with triethanolamine (1.5 microliters) added to maintain pH 8.0 for 14 hours at 37° C. with stirring.
Compound 1 thus formed was isolated by thin-layer chromatography on silica gel using a solvent mixture of chloroform/acetone/methanol/acetic acid/water (9:2:1.6:1:0.5 v:v). The compound was extracted with chloroform from the silica gel, dried and analyzed using High Resolution Mass Spectrometry (HRMS). The experimental mass achieved is 976.5899 which is in concurrence with the theoretical mass of 976.5917, as shown in
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Acetic Acid
Acetone
Anabolism
Chloroform
Diazomethane
Mass Spectrometry
Methanol
phosphoethanolamine
Silica Gel
Solvents
Thin Layer Chromatography
triethanolamine
All chemicals were obtained from commercial sources and were used without further purification. Fmoc- and Boc-protected amino acids were purchased from Iris Biotech GmbH (Marktredwitz, Germany), Sigma-Aldrich (Poznan, Poland), Bachem (Torrance, CA, USA), Creosalus (Louisville, KY, USA), and PE Biosciences Limited (Hong Kong, China). Fmoc-ACC-OH fluorescent dye was synthesized according to the procedure published previously by Maly et al.24 (link) Rink amide AM resin (200–300 mesh, loading 0.74 mmol g−1), 2-chlorotrityl chloride resin (100–200 mesh, loading 1.59 mmol g−1), biotin, HBTU, HATU, piperidine (PAP), diisopropylcarbodiimide (DICI), 2,2,2-trifluoroethanol (TFE), and trifluoroacetic acid (TFA) were purchased from Iris Biotech GmbH. Anhydrous HOBt was purchased from Creosalus. 2,4,6-Collidine (2,4,6-trimethylpyridine), acetonitrile (ACN, HPLC gradient grade), triisopropylsilane (TIPS), hydrobromic acid solution (30% HBr wt. in acetic acid), N-methylmorpholine (NMM), tetrahydrofuran (THF, anhydrous), isobutylchloroformate (IBCF), and 2,6-dimethylbenzoic acid (2,6-DMBA) were purchased from Sigma-Aldrich. N,N′-Dimethylformamide (DMF, pure for analysis), methanol (MeOH), dichloromethane (DCM), acetic acid (AcOH), diethyl ether (Et2O), and phosphorus pentoxide (P2O5) were obtained from POCh (Gliwice, Poland). Diazomethane used for the synthesis of AOMK inhibitors was generated according to the Aldrich Technical Bulletin (AL-180) protocol. All compounds (peptides, ACC fluorescent substrates, AIE fluorescent substrates, and inhibitors) were purified by reverse-phase HPLC on a waters system (Waters M600 solvent delivery module and waters M2489 detector system) using a semipreparative Discovery® C8 column (particle size 10 μm). The purity of the compounds was confirmed on the above HPLC system using an analytical Discovery® C8 column (particle size 10 μm). The solvent composition was as follows: phase A (water/0.1% TFA) and phase B (ACN/0.1% TFA). For purification and compound analysis, the assay was run for 30 min in a linear gradient (from 5% phase B to 100% phase B). The molecular weight of each compound was confirmed on a WATERS LCT Premier XE High Resolution Mass Spectrometer with electrospray ionization (ESI) and a time of flight (TOF) module. Antibodies for western blot analysis were purchased from R&D Systems.
1-hydroxybenzotriazole
2-chlorotrityl chloride
4-methylmorpholine
9,10-Dimethyl-1,2-benzanthracene
Acetic Acid
acetonitrile
Acids
Amides
Amino Acids
Anabolism
Antibodies
Biological Assay
Biotin
Diazomethane
Dimethylformamide
Ethyl Ether
Fluorescent Dyes
gamma-collidine
High-Performance Liquid Chromatographies
Hydrobromic acid
inhibitors
Iris Plant
Methanol
Methylene Chloride
Obstetric Delivery
Peptides
phosphoric anhydride
phosphorus pentoxide
piperidine
Resins, Plant
Solvents
tetrahydrofuran
Trifluoroacetic Acid
Trifluoroethanol
Western Blot
The detailed protocol for the synthesis of P1 Asp inhibitors and probes with AOMK electrophile warheads has recently been described by Poreba et al.26 (link) In brief, Boc-L-Asp(Bzl)-OH amino acids were transformed into Boc-L-Asp(Bzl)-CH2N2 through the use of diazomethane solution in diethyl ether. Next, the product was transformed into Boc-L-Asp(Bzl)-CH2Br with the use of a solution of HBr (30% wt. in acetic acid) and water. The crude product, a pale yellow oil, was then stirred with 2,6-dimethylbenzoic acid (2,6-DMBA) in the presence of KF in DMF to obtain Boc-L-Asp(Bzl)-AOMK. Then, the Boc group was removed using 50% TFA in DCM, and the final product (H2N-L-Asp(Bzl)-AOMK) was used for further synthesis without purification. In a separate synthesis, caspase-selective peptide fragments containing appropriate protecting groups (Ac-Glu(tBu)-Val-Glu(tBu)-Ile-COOH, Ac-hPhe-Val-Glu(tBu)-Ile-COOH and Ac-Val-Glu(tBu)-Ile-COOH) were synthesized on 2-chlorotrityl chloride resin and used without further purification. Next, the peptides were coupled with H2N-L-Asp(Bzl)-AOMK in DMF using HATU/DIPEA coupling reagents. The crude products were purified by HPLC, lyophilized and deprotected using 50% TFA in DCM, followed by removal of TFA/DCM under reduced pressure. Finally, the Bzl group from Asp was hydrolyzed via hydrogenolysis (Pd/C, H2, in DMF). The final products were purified by HPLC, lyophilized and dissolved in DMSO to a final concentration of 10 mM.
9,10-Dimethyl-1,2-benzanthracene
Acetic Acid
Acids
Amino Acids
Anabolism
Caspase
Chlorides
Diazomethane
DIPEA
Ethyl Ether
High-Performance Liquid Chromatographies
inhibitors
Peptide Fragments
Peptides
Pressure
Resins, Plant
Sulfoxide, Dimethyl
t-butyloxycarbonyl group
The recombinant LeAOS3 or ZmAOS (25 µg, 50 µg, 100 µg, 150 µg or 200 µg) dissolved in 100 mM phosphate buffer (100 µL), pH 7.0, was extensively vortexed with 9-HPOD, 13-HPOT or 13-HPOD (100 µg) in hexane (4 mL) at 0 °C for 20–60 s. The water was quickly frozen at −79 °C, and the hexane phase was decanted and treated with ethereal diazomethane at 0 °C for 3 min. The predominant part of hexane was evaporated in vacuo and an excess of ice-cold ethanol was directly added to the solution. After 30 min at 23 °C, solvent was evaporated, and the dry residue was treated with a trimethylsilylating mixture pyridine – hexamethyldisilazane – trimethylchlorosilane (2:1:2, by volume). The products were subjected to GC-MS analyses.
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9-hydroperoxy-10,12-octadecadienoic acid
13-hydroperoxy-9,11,15-octadecatrienoic acid
13-hydroperoxy-9,11-octadecadienoic acid
Buffers
Cold Temperature
Diazomethane
Ethanol
Ethyl Ether
Freezing
Gas Chromatography-Mass Spectrometry
hexamethyldisilazane
n-hexane
Phosphates
Pyridines
Solvents
trimethylchlorosilane
LeAOS3 and ZmAOS preparations (100 µg) dissolved in 100 mM phosphate buffer (100 µL), pH 7.0, were extensively vortexed with 9-HPOD (200 µg) in hexane (4 mL) at 0 °C for 60 s. The water was quickly frozen at −79 °C, and the hexane phase was decanted. The resulting allene oxide (free acid) preparation was allowed to stay in hexane solution for 20 h. Then, the products were treated with diazomethane, trimethylsilylated and subjected to GC-MS analyses.
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9-hydroperoxy-10,12-octadecadienoic acid
Acids
Buffers
Diazomethane
Freezing
Gas Chromatography-Mass Spectrometry
n-hexane
Oxides
Phosphates
propadiene
Top products related to «Diazomethane»
Sourced in Germany
Silica gel 60H is a type of adsorbent material used in various laboratory applications. It is a porous, inert substance made of amorphous silicon dioxide. Silica gel 60H has a high surface area and is commonly used for adsorption, filtration, and drying processes in laboratories.
Sourced in France, Germany
Trimethylsilyldiazomethane is a reagent used in organic synthesis for the methylation of carboxylic acids and the formation of silyl esters. It functions as a methylating agent.
Sourced in United States, Germany, Canada, Japan, Spain
The 6890N Network GC System is a gas chromatography instrument designed for analytical laboratory applications. It features a network-ready design for remote access and control. The system is capable of performing gas chromatographic separations and analysis of various chemical samples.
Sourced in United States
The 5973 Network Mass Selective Detector is a lab equipment product designed to perform mass spectrometric analysis. It functions as a detector for gas chromatography systems, providing precise identification and quantification of chemical compounds.
NHS-diazirine is a heterobifunctional crosslinking reagent used for covalent protein-protein and protein-ligand conjugation. It contains an N-hydroxysuccinimide (NHS) ester group for primary amine labeling and a photo-activatable diazirine group for non-specific photo-crosslinking.
DEAE cellulose column is a chromatography medium used for the purification and separation of biomolecules. It is composed of diethylaminoethyl (DEAE) functional groups attached to cellulose matrix. The DEAE groups provide a positively charged surface that can interact with negatively charged molecules, allowing for the separation and purification of various biomolecules, such as proteins, nucleic acids, and enzymes.
Sourced in United Kingdom
Lab-Base is a high-quality laboratory workstation designed to provide a stable and organized work environment for various scientific applications. It offers a durable, scratch-resistant surface and integrated storage options to optimize workspace efficiency.
Sourced in United States, Germany, Japan
The Agilent 6890N Gas Chromatograph is an analytical instrument designed for separating and identifying the individual components of a complex chemical mixture. It operates by heating the sample and using an inert carrier gas to transport the vaporized components through a column, where they are separated based on their different boiling points and interactions with the column material. The separated components are then detected and identified using various detectors, such as flame ionization or mass spectrometry.
Sourced in Germany
Sulfo-LC-SDA is a water-soluble, amine-reactive biotinylation reagent used for protein labeling. It selectively modifies primary amines on proteins and peptides, allowing for subsequent detection or purification via streptavidin or avidin binding.
TMS-diazomethane is a laboratory reagent used in organic synthesis. It is a source of the diazomethyl group, which can be used in various chemical reactions. The core function of TMS-diazomethane is to serve as a methylating agent in organic chemistry.
More about "Diazomethane"
Diazomethane is a highly reactive organic compound that plays a crucial role in various chemical reactions, particularly in the synthesis of complex organic molecules.
This powerful methylating agent is widely used in the preparation of esters, ethers, and other derivatives, making it an invaluable tool for chemists investigating intricate molecular structures.
Researchers can leverage advanced tools like PubCompare.ai to optimize their diazomethane-based studies.
This innovative platform allows them to easily locate relevant protocols from literature, preprints, and patents, and then use AI-driven comparisons to identify the most accurate and reproducible methods.
This can help enhance the accuracy and reproducibility of diazomethane-related research, leading to more reliable and impactful findings.
Diazomethane's diverse applications extend beyond organic chemistry, with related compounds like Trimethylsilyldiazomethane and NHS-diazirine also playing crucial roles in various scientific endeavors.
Effiicient purification techniques, such as DEAE cellulose column chromatography and Silica gel 60H, can be employed to isolate and purify diazomethane and its derivatives.
Advanced analytical instruments, such as the 6890N Network GC System and the 5973 Network Mass Selective Detector, can be utilized to characterize and analyze the products of diazomethane-mediated reactions.
Additionally, Lab-Base and other software tools can assist in streamlining the data management and analysis processes, further enhancing the efficiency and productivity of diazomethane-based research.
By leveraging the insights and tools available, researchers can navigate the complexities of diazomethane-related studies with greater ease and confidence, ultimately driving advancements in the field of organic chemistry and beyond.
This powerful methylating agent is widely used in the preparation of esters, ethers, and other derivatives, making it an invaluable tool for chemists investigating intricate molecular structures.
Researchers can leverage advanced tools like PubCompare.ai to optimize their diazomethane-based studies.
This innovative platform allows them to easily locate relevant protocols from literature, preprints, and patents, and then use AI-driven comparisons to identify the most accurate and reproducible methods.
This can help enhance the accuracy and reproducibility of diazomethane-related research, leading to more reliable and impactful findings.
Diazomethane's diverse applications extend beyond organic chemistry, with related compounds like Trimethylsilyldiazomethane and NHS-diazirine also playing crucial roles in various scientific endeavors.
Effiicient purification techniques, such as DEAE cellulose column chromatography and Silica gel 60H, can be employed to isolate and purify diazomethane and its derivatives.
Advanced analytical instruments, such as the 6890N Network GC System and the 5973 Network Mass Selective Detector, can be utilized to characterize and analyze the products of diazomethane-mediated reactions.
Additionally, Lab-Base and other software tools can assist in streamlining the data management and analysis processes, further enhancing the efficiency and productivity of diazomethane-based research.
By leveraging the insights and tools available, researchers can navigate the complexities of diazomethane-related studies with greater ease and confidence, ultimately driving advancements in the field of organic chemistry and beyond.