Full details of the acylcarnitine preparation are described elsewhere (19 (link)). Briefly, aliquots of the skeletal muscle raw extract were transferred to tubes, dried by SpeedVac, and reconstituted in 0.1% phosphoric acid. Acylcarnitines were then loaded onto Oasis MCX extraction cartridges (Waters Corp.) and activated by methanol immediately before sample loading. Cartridges were washed twice with 0.1% phosphoric acid before samples were eluted with methanol. Eluates were dried by SpeedVac, reconstituted in mobile phase (as described below), and transferred to a vial for chromatography.
Oasis mcx extraction cartridges
Manufactured by Waters Corporation
Sourced in United States
The Oasis MCX extraction cartridges are solid-phase extraction (SPE) products designed for the purification and concentration of analytes from complex matrices. These cartridges utilize a mixed-mode cation exchange sorbent to selectively retain and separate compounds based on their ionic and hydrophobic properties.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Speedvac, by Thermo Fisher Scientific (1 mentions)
Trypsin, by Promega (1 mentions)
Direct q 3 uv, by Merck Group (1 mentions)
4 7 8 trimeiqx, by LGC (1 mentions)
Meaαc, by LGC (1 mentions)
Meiqx, by LGC (1 mentions)
4 8 dimeiqx, by LGC (1 mentions)
7 8 dimeiqx, by LGC (1 mentions)
Empower software, by Waters Corporation (1 mentions)
4 protocols using oasis mcx extraction cartridges
1
Preparation of Acylcarnitines from Skeletal Muscle
2
Proteomic Workflow for Quantitative Analysis
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Precipitated proteins were washed with acetone and dried. The pellets were dissolved in 300 µL of 8 M urea and processed by the filter aided sample preparation method [15 (link)] using Vivacon 10 kDa MWCO filter (Sartorius Stedim Biotech). Proteins were washed twice with 100 µL of 8 M urea and reduced by 100 µL of 10 mM DTT. After reduction, proteins were incubated with 100 µL of 50 mM IAA and washed twice with 100 µL of 25 mM TEAB. Trypsin (Promega) was used at 1:50 ratio (w/w) and the digestion proceeded for 16 h at 30 °C.
For comparative analysis, peptide concentration was determined spectrophotometrically (Nanodrop, Thermo Scientific) and samples from the same group of chickens were pooled. Pooled samples were then labelled using the stable isotope dimethyl labelling protocol as described previously [16 (link)]. Labeled samples were mixed and 3 subfractions were prepared using Oasis MCX Extraction Cartridges (Waters). The samples were desalted on SPE C18 Extraction Cartridges (Empore) and concentrated in a SpeedVac (Thermo Scientific) prior to LC–MS/MS.
For comparative analysis, peptide concentration was determined spectrophotometrically (Nanodrop, Thermo Scientific) and samples from the same group of chickens were pooled. Pooled samples were then labelled using the stable isotope dimethyl labelling protocol as described previously [16 (link)]. Labeled samples were mixed and 3 subfractions were prepared using Oasis MCX Extraction Cartridges (Waters). The samples were desalted on SPE C18 Extraction Cartridges (Empore) and concentrated in a SpeedVac (Thermo Scientific) prior to LC–MS/MS.
3
Analysis of Heterocyclic Aromatic Amines
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All chemicals and solvents used for HAA analysis were HPLC-grade and procured from Merck Co. (Darmstadt, Germany). Ultra-pure water was obtained from a Milli-Q water purification system (Millipore, Direct Q@3 UV). Oasis MCX extraction cartridges (3 cm3/60 mg) were secured from Waters (Milford, MA, USA). The standards of HAAs including IQx, IQ, MeIQx, MeIQ, 7,8-DiMeIQx, 4,8-DiMeIQx, 4,7,8-TriMeIQx, PhIP, AαC, and MeAαC were acquired from Toronto Research Chemicals (Downsview, Ontario, ON, Canada). 4,7,8-TriMeIQx was used as an internal standard. Standard stock solutions in methanol were prepared as described by Oz [22 (link)].
4
HPLC Analysis of TRP Metabolites
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The TRP metabolites were measured by a previously described HPLC method [22 (link)]. Briefly, analytes were extracted from samples and calibrators/controls using Waters Oasis MCX extraction cartridges (Waters, Milford, MS). The eluent was then evaporated to dryness and reconstituted with 0.1 M PBS for injection into the HPLC system. Analyses were carried out on a Waters 2695 chromatograph with a 250 mm × 4 mm Supersphere 60 RP-select B, C8 column (Merck, Darmstadt, Germany) connected to a Waters 2487 dual-λ UV detector and a 2475 fluorescence detector. TRP (λex: 300 nm; λem: 350 nm) and 5-HIAA (λex: 300 nm; λem: 340 nm) were measured by fluorescence detection; KYN (365 nm), KYNA (330 nm), and 3-HK (365 nm) were measured by UV detection. Data were processed using EMPOWER software (Waters). The concentrations were established through comparison of peak heights of the single analytes with the peak heights of the respective calibration curves, always including the internal standard into the calculation. The method has been validated showing good absolute recovery and precisions (e.g. for 3-HK: recovery of 85,8%, intra-day precision of 3.9%, and inter-day precision of 7.5%).
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