On day 1, a total of 300,000 human VSMCs were plated on 25 cm2 culture flasks (Thermo Fisher Scientific). On the next day, the culture medium was changed into a new one which contained 1, 5, or 10 mmol/L of metformin. After 48 hours, the cells were analysed for UDP-sugars as previously described.27 (link) Briefly, the cells were placed on ice and washed with cold PBS, where after they were scraped into cold PBS, followed by sonication using SONOPULS (Bandelin, Berlin, Germany). After sonication, small aliquots were collected and their total protein content was measured with Pierce BSA protein assay kit (Thermo Scientific). Total protein amounts were used to normalise the UDP-sugar results. The rest of the solution was centrifuged (6000×g for 20 minutes), and further purified with Supelclean Envi-Carb SPE tubes (Sigma, Saint Louis, MO) as previously described.28 (link) Subsequently, the eluted samples were evaporated by vacuum centrifugation and dissolved in water for anion exchange high-performance liquid chromatography with a CarpoPac PA1 column (Dionex, Thermo Fisher Scientific) as previously described.27 (link)
Supelclean envi carb spe tubes
Manufactured by Merck Group
Sourced in United States
Supelclean™ ENVI™-Carb SPE Tubes are solid-phase extraction (SPE) devices designed for the purification and concentration of organic compounds from various sample matrices. The tubes contain a porous, graphitized carbon sorbent material that selectively retains organic analytes, allowing for the removal of interfering substances. These tubes are intended for use in analytical sample preparation workflows.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (3 mentions)
Milli q system, by Merck Group (2 mentions)
25 cm2 culture flasks, by Thermo Fisher Scientific (1 mentions)
Pierce bsa protein assay kit, by Thermo Fisher Scientific (1 mentions)
Sonopuls, by Bandelin (1 mentions)
Hplc grade acetonitrile, by Merck Group (1 mentions)
Chloroform, by Merck Group (1 mentions)
Concentrator 5301, by Eppendorf (1 mentions)
Milli q plus ultrapure water system, by Merck Group (1 mentions)
Analytical grade acetone, by Merck Group (1 mentions)
Na2so4, by Thermo Fisher Scientific (1 mentions)
Sepra c18 e, by Phenomenex (1 mentions)
Toluene, by Merck Group (1 mentions)
Formic acid, by Merck Group (1 mentions)
Sulphuric acid, by Merck Group (1 mentions)
8 protocols using supelclean envi carb spe tubes
1
Metformin Modulates UDP-Sugar Levels in VSMCs
2
Quantification of UDP-Sugars by HPLC
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Deionized water was prepared by a Milli-Q System (Millipore Corporation, Billerica, USA). HPLC-grade acetonitrile was purchased from Sigma-Aldrich (Steinheim, Germany). Standards of uridine 5′-diphosphate disodium salt hydrate (UDP, ≥96.0 %), uridine 5′-diphosphate-glucose (UDP-Glc), uridine 5′-diphosphate-galactose (UDP-Gal), uridine 5′-diphosphate-glucuronic acid (UDP-GlcA), were purchased from Sigma-Aldrich (Steinheim, Germany). Uridine 5′-diphosphate-arabinose (UDP-Ara), uridine 5′-diphosphate-xylose (UDP-Xyl) and uridine 5′-diphosphate-galacturonic acid (UDP-GalA) were from Carbosource (University of Georgia, Athens, GA, USA). UDP-sugars were quantified by UV absorbance on a Nanodrop ND-1000 spectrophotometer (NanoDrop Products, Thermo Scientific, Wilmington, DE, USA), using UDP as reference. Trifluoroacetic acid (≥99.5 %), HPLC grade methanol, and 2-propanol were purchased from Fluka Analytical (Buchs, Switzerland). Ammonia (25 %) and chloroform (≥99.0–99.4 %) were purchased from Merck (Darmstadt, Germany). Peek capillaries of various sizes as well as nuts, ferrules, sleeves, finger tights, and metal unions were purchased from Upchurch Scientific (Oak Harbor, USA). Supelclean ENVI-Carb SPE Tubes (3 ml, 0.25 g, particle size: 120–400 mesh) were obtained from Sigma-Aldrich. Sample vials, vial inlets, and vial snapcaps were purchased from VWR International (Radnor, USA).
3
Okra Glycoprotein N-Glycan Release and Analysis
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N-glycans with PNGase A were released from the okra glycoprotein using the previously described method by Triguero et al. (2010) . The okra glycoprotein sample (0.5 g) was digested with 0.64 g of pepsin in 150 mL of HCl (pH 2.0) at 37°C for 16 h, and the pH was adjusted to 2.0 every half hour. The solution was boiled for 10 min to deactivate the enzyme, and the reaction mixture was further lyophilised. Following lyophilisation, the sample was deglycosylated with PNGaseA in 0.1 M citric acid buffer (pH 5.0) at 37°C for 48 h. Then, the mixture was heated for 5 min at 100°C to deactivate the enzyme, and then freeze-dried.
The method of releasing N-glycans by PNGase F from the okra glycoprotein has been previously described by Zhao et al. (2018a) . Briefly, 1.5 mg of the okra glycoprotein was added to 8 M urea and 10 mM dithiothreitol to denature the glycoprotein. After that, the reaction solution was washed with 40 mM NH 4 HCO 3 , and further digested with 2 μL of PNGase F at 37°C for 12 h. The N-glycans of the okra glycoprotein were collected with Supelclean ENVI-Carb SPE tubes (Sigma-Aldrich, Inc., Bellefonte, PA), and lyophilised. The reaction mixture released from PNGase A was mixed with the reaction mixture released from PNGase F, and detected by MALDI-TOF-MS.
The method of releasing N-glycans by PNGase F from the okra glycoprotein has been previously described by Zhao et al. (2018a) . Briefly, 1.5 mg of the okra glycoprotein was added to 8 M urea and 10 mM dithiothreitol to denature the glycoprotein. After that, the reaction solution was washed with 40 mM NH 4 HCO 3 , and further digested with 2 μL of PNGase F at 37°C for 12 h. The N-glycans of the okra glycoprotein were collected with Supelclean ENVI-Carb SPE tubes (Sigma-Aldrich, Inc., Bellefonte, PA), and lyophilised. The reaction mixture released from PNGase A was mixed with the reaction mixture released from PNGase F, and detected by MALDI-TOF-MS.
4
Shellfish Toxin Extraction Protocol
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TTX extraction was carried out following the conditions described in [28 (link)] with slight modifications as mentioned in the previous section. Briefly, 5 g of shellfish homogenate were weighed into a 50 mL polypropylene centrifuge tube. 5 mL of 0.1% acetic acid (LC-MS grade, Fluka Analytical, Steinheim, Germany) was added to the sample and homogenized by vortex for 90 s. the centrifuge tube was capped and placed in a boiling water bath for 5 min, then cooled to room temperature and vortex-mixed for 90 s. The sample was centrifuged at 4000× g for 10 min., 1 mL of supernatant was transferred to a 1.5 mL centrifuge tube, and 5 µL of 25% ammonium hydroxide (LC-MS grade, Fluka Analytical, Steinheim, Germany) was added, vortexed, and centrifuged at 10,000× g for 60s for further clean up through graphitized carbon SPE cartridges (Supelclean ENVI CARB SPE tubes), 3 mL (0.25 g) from SUPELCO (Bellfonte, PA, USA) before HILIC-MS/MS analysis.
5
Extraction and Purification of UDP-Sugars
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Samples were applied to Supelclean™ ENVI™-Carb SPE Tubes (3 ml, 0.25 g, particle size: 120–400 mesh) (Supelco) equilibrated with 3 ml equilibration-elution buffer (60% acetonitrile in water containing 0.3% formic acid adjusted to pH 9 with ammonia) and flushing with 3 ml of water. After sample application, washing steps were performed, first with 3 ml of water and afterwards with 1 ml wash buffer (60% acetonitrile in water). Sample elution was done by flushing the column with 2 ml equilibration-elution buffer [29] (link). The aqueous phase containing the UDP-sugars was vaporized by under vacuum in a concentrator 5301 (Eppendorf) at 30°C and reconstituted in 100 µl water.
6
Pesticide Analytical Standards Preparation
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Pesticide analytical standards were purchased from Chemservice (West Chester, PA) and Sigma-Aldrich Canada (Oakville, ON). Analytical grade acetone and toluene were purchased from EMD Millipore (Darmstadt, Germany). Analytical grade acetonitrile and Na2SO4 were purchased from Fisher Scientific (Fairlawn, NJ). Water was obtained from a Milli-Q® Plus Ultra Pure Water system (Millipore Corp., Burlington, MA). Sepra™C18-E was obtained from Phenomenex (Torrance, CA). Supelclean™ ENVI™-Carb SPE Tubes were obtained from Supelco (Bellefonte, PA). Sep-Pak® Classic NH2 Cartridges were obtained from Waters Corp. (Milford, MA).
7
Quantitative Analysis of Pyrrolizidine Alkaloids
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Methanol and acetonitrile were HPLC grade and purchased from Biosolve BV (Valkenswaard, the Netherlands). Water was purified using a Millipore Milli-Q system (Millipore Corp., Bedford, MA, USA). Formic acid (98–100%) and sulphuric acid (95–97%) were ordered from Merck (Kenilworth, New Jersey, USA). Supelclean™ ENVI™-Carb SPE tubes 500 mg/6 mL (Supelco, Bellefonte, Pennsylvania, USA) were obtained from Sigma-Aldrich and Strata™-XC 33 µm Polymeric Strong cation exchange SPE cartridges, 500 mg/6 mL were bought from Phenomenex®.
Standards of pyrrolizidine alkaloids (PAs) and related N-oxides (PANOs) were ordered from PhytoLab GmbH and Co. KG (Germany): 16 PAs i.e. echimidine, erucifoline, europine, heliotrine, indicine, intermedine, jacobine, lasiocarpine, lycopsamine, monocrotaline, retrorsine, senecionine, seneciphylline, senecivernine, senkirkine, trichodesmine and 14 PANOs i.e. echimidine N-oxide, erucifoline N-oxide, europine N-oxide, heliotrine N-oxide, indicine N-oxide, intermedine N-oxide, jacobine N-oxide, lasiocarpine N-oxide, lycopsamine N-oxide, monocrotaline N-oxide, retrorsine N-oxide, senecionine N-oxide, seneciphylline N-oxide, senecivernine N-oxide. Purity was between 89% and 100%. Individual solutions of each compound at the concentration of 1 mg/mL were prepared in methanol and have proven to be stable for 1 year at −20 °C.
Standards of pyrrolizidine alkaloids (PAs) and related N-oxides (PANOs) were ordered from PhytoLab GmbH and Co. KG (Germany): 16 PAs i.e. echimidine, erucifoline, europine, heliotrine, indicine, intermedine, jacobine, lasiocarpine, lycopsamine, monocrotaline, retrorsine, senecionine, seneciphylline, senecivernine, senkirkine, trichodesmine and 14 PANOs i.e. echimidine N-oxide, erucifoline N-oxide, europine N-oxide, heliotrine N-oxide, indicine N-oxide, intermedine N-oxide, jacobine N-oxide, lasiocarpine N-oxide, lycopsamine N-oxide, monocrotaline N-oxide, retrorsine N-oxide, senecionine N-oxide, seneciphylline N-oxide, senecivernine N-oxide. Purity was between 89% and 100%. Individual solutions of each compound at the concentration of 1 mg/mL were prepared in methanol and have proven to be stable for 1 year at −20 °C.
8
Xyloglucan Oligosaccharide Analysis in Arabidopsis
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Alcohol‐insoluble residue (AIR) was prepared from leaf tissue or etiolated hypocotyls of Col‐0, Atmur3.1 xlt2, and generated Arabidopsis complementation lines by grinding dried plant material followed by an extraction with 70% (v/v) aqueous ethanol and twice with chloroform:methanol (1:1) (Schultink et al., 2013 (link)). The AIR material was then subjected to a xyloglucanase (XEG) digestion (Jensen et al., 2012 (link); Pauly et al., 1999 (link)). The resulting solubilized XyG oligosaccharides were desalted by solid‐phase extraction using Supelclean™ ENVI‐Carb™ SPE Tubes (Supelco, 57109‐U) (Schultink et al., 2013 (link); Zhu et al., 2018 (link)) and analyzed by MALDI‐TOF mass spectrometry and High‐performance anion‐exchange chromatography (HPAEC) equipped with a pulsed amperometric detector (PAD; IC Amperometric Detector, Metrohm) as described (Schultink et al., 2013 (link)). XyG oligosaccharides were separated using the following gradient: flow .4 ml/min, 100 mM NaOH to 100 mM NaOH/80 mM sodiumacetate in 22 min followed by a return to 100 mM NaOH within 4 min and a 6 min 100 mM NaOH wash. The XyG oligosaccharide mixture was also subjected to acid hydrolysis followed by monosaccharide compositional analysis by HPAEC as described (Jensen et al., 2012 (link)).
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