PPC and AMG tissues were extracted with 500 µl of Chilled extraction solvent (acetonitrile:methanol:water, 2:2:1, v/v) containing 5 µM ATP-13C10 (Sigma-Aldrich), which was used as an internal standard. HPC tissue was extracted with 800 µl of the same extraction solvent with the internal standard. The mixtures were sonicated for about 7 min (10 cycles), vortexed for 10 min, and then incubated at − 20 °C for 1 h. The samples were centrifuged at 18,000×g for 10 min at 4 °C. The supernatants were collected and stored at − 20 °C. The pellets were re-extracted using 200 µl aliquots of the extraction solvent and the procedure was repeated. The supernatants were combined and dried under nitrogen flow in a nitrogen evaporator. The dry extracts were reconstituted with 100 µl of 60% acetonitrile (ACN) and filtered through a 0.22 µm PTFE filter before analysis.
Atp 13c10
Manufactured by Merck Group
Sourced in United Kingdom
The ATP-13C10 is a laboratory equipment product designed for scientific research. It is a high-precision instrument used for the detection and analysis of carbon-13 labeled adenosine triphosphate (ATP) molecules. The core function of the ATP-13C10 is to facilitate the quantitative measurement of cellular energy metabolism and related biochemical processes.
Automatically generated - may contain errors
Lab products found in correlation
Methanol and water, by Thermo Fisher Scientific (1 mentions)
Amp15n5, by Merck Group (1 mentions)
Ammonium hydroxide, by Merck Group (1 mentions)
Bca assay, by Thermo Fisher Scientific (1 mentions)
D glucose 13c6, by Merck Group (1 mentions)
15n l leucine, by Merck Group (1 mentions)
Anti ha magnetic beads, by Thermo Fisher Scientific (1 mentions)
4 protocols using atp 13c10
1
Metabolite Extraction from PPC, AMG, and HPC Tissues
2
HILIC Analysis of Metabolite Standards
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All solvents and mobile phase additives were LC-MS-grade. Acetonitrile (CHROMASOLV LC-MS) was purchased from Scientific Laboratory Supplies (Wilford, UK), methanol and water were purchased from Fisher Scientific (Leicestershire, UK), ammonium bicarbonate was obtained from Fluorochem (Hadfield, UK), and ammonium hydroxide was from Sigma-Aldrich (Gillingham, UK). All authentic chemical standards used for the annotation of metabolites were of analytical or higher grade and were obtained from various suppliers (Sigma-Aldrich (Gillingham, UK), VWR (Leicestershire, UK), and Fisher Scientific (Leicestershire, UK)). They were accurately weighed and dissolved in appropriate volumes of LC-MS-grade water to obtain individual stock solutions at concentrations of 1 mg/mL. Appropriate amounts of each stock solution were mixed to obtain four standard mixtures at concentrations of 25 µg/mL in water that were further prepared for HILIC analysis, as described below. Stable isotope-labelled internal standards AMP-15N5, ADP-15N5, and ATP-13C10 were purchased from Sigma-Aldrich (Gillingham, UK) and prepared in a mixture at a concentration of 48 µg/mL.
3
Metabolite Extraction and Isotope Tracing Protocol
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Cells on six-well plates were washed twice with the wash buffer (75 mM ammonium carbonate, pH 7.4) and the plates were flash-frozen in liquid nitrogen. 800 μl extraction buffer (acetonitrile:methanol:H2O = 4:4:2, −20°C) was added to the wells, scraped, and centrifuged by 21,000g for 20 min at 4°C. The supernatants were dried by vacuum centrifugation (Labogene) for 6 h at 20°C. Pellets were lysed in 50 mM Tris-KOH pH 8.0, 150 mM NaCl, 1% SDS, and used for protein quantification using the BCA assay (23225; Thermo Fisher Scientific). To measure steady-state levels of metabolites, the following internal standards were added to the extraction buffer: 2.5 mM amino acid standard (MSK-A2-1.2; CIL), 100 μg/ml citrate d4 (485438; Sigma-Aldrich), 1 mg/ml 13C10 ATP (710695; Sigma-Aldrich). No internal standard was added for the stable isotope-tracing experiments. Isotopologues used in the experiments are as follows: 13C6 D-glucose (389374; Sigma-Aldrich), 2,3,3-2H3 L-serine (DLM-582; CIL), 13C6 L-leucine (605239; Sigma-Aldrich), 15N L-leucine (340960; sigma-Aldrich), 13C5 L-valine (758159; Sigma-Aldrich), 15N L-valine (490172; Sigma-Aldrich). Isotopologues were added to the regular culture medium (MEM supplemented with 9.5% undialyzed FBS) and treated to cells as indicated in each figure legend.
4
Mitochondrial Metabolite Extraction Protocol
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Relative metabolite levels were largely determined as described previously31 (link). In brief, 30 × 106 HA-MITO-expressing MEFs were plated one day before the experiment and all subsequent steps were performed at 4 °C. Cells were washed twice and collected in KPBS (136 mM KCl, 10 mM KH2PO4–KOH pH 7.25) before centrifugation at 1,000g for 2 min. Cells were resuspended in KPBS and homogenized with 15 strokes using a glass Teflon homogenizer at 1000 rpm. The homogenates were centrifuged at 1,000g for 3 min and the supernatant was incubated with magnetic anti-HA beads (Thermo Fisher Scientific) on an end-over-end rotator for 3 min at 15 r.p.m. Beads were collected on a magnet and washed four times with KPBS. Mitochondrial metabolites were extracted by incubating the beads with extraction buffer (40:40:20 v/v/v acetonitrile/methanol/water containing 10 ng ml−1 of 13C10-ATP (Sigma) as an internal standard) for 5 min. For each experiment, an input and IP fraction were taken for immunoblot analysis of mitochondrial enrichment and purity. The organellar-specific anionic metabolites were analysed as described in the whole-cell metabolite section. Data was normalized to the succinate dehydrogenase complex flavoprotein subunit A (SDHA) protein level determined for each IP fraction by immunoblot densitometry.
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