The 5 ml of blood was drawn on the EDTA as an anticoagulant, centrifuged and the plasma was separated. Plasma proteins from 10 μl of plasma were reduced by adding dithiotreitol (DTT) (Merck, Darmstadt, Germany) to final concentration of 0.5 M, followed by 100 mM iodoacetamide (IAA) (Sigma-Aldrich, St. Louis, MO, USA) alkylation. Denaturated glycoproteins were incorporated in SDS-polyacrylamide gel blocks to minimize the possibility of renaturation and glycoprotein loss. Gels were alternately washed with 100% acetonitrile (J.T. Baker, Phillipsburg, NJ, USA) and 20 mM NaHCO3 (Merck, Darmstadt, Germany) buffer causing continuous dehydration and hydration of the gels, respectively, to remove all free detergents, reducing and alkylating agents. The enzyme, N-glycosidase-F (PNGaseF, Prozyme, Hayward, CA, USA) was absorbed by gel blocks and used to release the N-glycans from the proteins. Glycans were removed from gels by alternate washing with water and 100% acetonitrile, collected in a 96-well collection plate (Waters, Milford, MA, USA) and dried in a vacuum centrifuge (Thermo Scientific, Waltham, MA, USA)52 (link).
96 well collection plate
Manufactured by Waters Corporation
Sourced in United States
The 96-well collection plate is a laboratory equipment designed for sample collection and storage. It features a grid of 96 individual wells, each capable of holding a small volume of liquid sample. The plate is commonly used in high-throughput applications, such as sample preparation and assay development, to facilitate the efficient handling and organization of multiple samples simultaneously.
Automatically generated - may contain errors
Lab products found in correlation
Iodoacetamide iaa, by Merck Group (1 mentions)
Acetonitrile, by Avantor (1 mentions)
Nahco3, by Merck Group (1 mentions)
Vacuum centrifuge, by Thermo Fisher Scientific (1 mentions)
Amicon ultra 15, by Merck Group (1 mentions)
Captiva 0.45 μm protein precipitation filtration plate, by Agilent Technologies (1 mentions)
7 protocols using 96 well collection plate
1
Plasma N-Glycan Profiling Protocol
2
Plasma Sample Preparation for Analyte Quantification
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Filtrate samples were pretreated by protein precipitation with 50% MeOH and acetonitrile. Blank filtrate was prepared using plasma obtained from healthy volunteers. Plasma sample was centrifuged in an Amicon® Ultra‐15 centrifugal filter device (Merk Millipore Ltd.) at 20,600 × g at 4°C until almost all the plasma was filtered, and the supernatant was collected. For calibrator and QC samples, 50 μl of blank filtrate was transferred to a 2‐ml polypropylene tube, and 50 μl of each calibrator or QC sample in 50% MeOH, 25 μl of IS working solution in 50% MeOH, and 100 μl of acetonitrile were added in that order. For patient filtrate and blank filtrate samples, 50 μl of filtrate sample, 25 μl of IS solution in 50% MeOH, 100 μl of acetonitrile, and 50 μl of 50% methanol were added in that order into a polypropylene tube. The mixtures were vortexed for 1 min, and centrifuged at 20,600 × g at 4°C for 10 min. The supernatants were collected and transferred to a 96‐well collection plate (Waters Corp.). The sample plate was sealed with a sealing cap (Waters Corp.) and kept at 4°C until assay. Twenty microliters of sample was injected into the UHPLC.
3
Sensitive Plasma and Brain Tissue Bioanalysis
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For QC and calibrator samples, the respective working solutions (25 µL) were mixed with blank plasma (100 µL) or blank tissue matrix (100 µL; 40 mg/mL brain tissue in water), whereas, for analytical samples 100 µL of plasma or 100 µL of brain solution (40 mg/mL brain tissue in water) was used, and 25 µL of ACN/water (1/1, v/v) was added for volume compensation. All samples except blanks were mixed with 25 µL of IS working solution. For the purpose of liquid–liquid extraction, 100 µL of 0.2 M borate buffer (pH 9) and 2 mL TBME were added to each sample. After 10 min of overhead shaking and subsequent centrifugation (10 min, 3000× g, 15 °C), 25 µL (high concentration assay) or 1.5 mL (low concentration assay, brain tissue assay) of the organic phase was transferred to a glass tube and evaporated to dryness under a heated stream of nitrogen (10 min, 40 °C). The residue was dissolved in 500 µL (high concentration assay, brain tissue assay) or 100 µL (low concentration assay) eluent (water with 9.75% ACN and 0.1% FA) in an ultrasonic device for 1 min and transferred to a 96-well collection plate (Waters Corporation, Milford, MA, USA) for measurement.
4
Plasma and Brain Tissue Protein Extraction
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For protein precipitation of plasma microsamples, an excess of ACN (50 µL) with 0.1% FA containing the IS was applied to an Impact® 96-well protein precipitation plate (Phenomenex, Torrance, CA, USA). Subsequently, 20 µL of the plasma samples were added and the plates sealed and shaken for 1 min. Brain tissue homogenate samples were processed accordingly. For protein depletion, samples were filtered into a 96-well collection plate (Waters, Milford, MA, USA) by applying positive pressure with a 96-well positive pressure unit (Waters, Milford, MA, USA) operated with air overpressure of 5 psi. After addition of 40 µL of water containing 0.1% FA, the collection plates were sealed, shaken, and extracts injected onto the UPLC system.
5
Quantification of NHC and NHCtp in Biological Matrices
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For quantification of NHC in human plasma, sample preparation was carried out via protein precipitation using a 96-well Captiva 0.45 μm protein precipitation filtration plate (Agilent Technologies, Santa Clara, CA). Briefly, 50 µL of sample was pipetted into the Captiva plate wells, followed by 50 µL of NHC-IS solution. Next, 0.5 mL of acetonitrile was added to each well and samples were incubated for 5 min. Samples were eluted via vacuum filtration, evaporated to dryness, and reconstituted in 0.6 mL of 0.1% formic acid.
For quantification of NHCtp in PBMC lysate, 100 µL of sample was pipetted into a 96-well collection plate (Waters Corporation, Milford, MA), followed by 25 µL of NHCtp-IS. Samples were evaporated to dryness under nitrogen, and reconstituted in 100 µL water.
For quantification of NHCtp in PBMC lysate, 100 µL of sample was pipetted into a 96-well collection plate (Waters Corporation, Milford, MA), followed by 25 µL of NHCtp-IS. Samples were evaporated to dryness under nitrogen, and reconstituted in 100 µL water.
6
Calibration and QC Sample Preparation
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Blood samples for calibration and QC purposes were generated through the addition of 25 µL of the pertinent spike solution to 20 µL of blank blood and the subsequent addition of 25 µL of IS solution. Calibration samples were produced at 10, 30, 100, 300, 1000, 3000, and 10,000 ng/mL, and QC samples were produced at concentrations of 10, 30, 3750, and 7500 ng/mL. For study sample processing, 20 µL of each sample was added to 25 µL of IS solution, and 25 µL of ACN/H2O 1/1 + 0.1% FA was added for volume compensation to ensure that sample preparation matches that of study blood samples. For stability reasons, each sample was immediately depleted from proteins by adding 150 µL ACN including 0.1% FA. Samples were processed individually and kept frozen until processing. Subsequently, samples were centrifuged at 13,200× g for 5 min. From the extracts, 10 µL was transferred to 400 µL of ACN/H2O 1/19 + 0.1% FA in wells of a 96-well collection plate (Waters, Milford, MA, USA).
7
Urine Sample Preparation for Analysis
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Twenty-five microliters of urine sample or QC are mixed with 250 µL of the IS working solution in a 1 mL well from a 96-well filtration plate (AcropreoAdv 0.2 µm WWPTFE, Pall Corporation, Port Washington, NY, USA). Thereafter, the samples are filtrated using a centrifuge (Beckman Coulter, Model, J-E, Brea, CA, USA) at 829× g for 2 min, and retrieved in a 1 mL 96-well collection plate (Waters Corp.).
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