To generate samples for proteomic analysis, the frozen biological samples were pulverized in a mortar containing liquid nitrogen to prevent thawing. The resulting powder was solubilised in 330 μl lysis buffer (2 mol/l Thiourea, 6 mol/l Urea, 4% CHAPS, 1 cOmplete ULTRA Tablets Mini (Roche, Penzberg, Germany) per 5 ml buffer). Afterwards, each sample was centrifuged using a QIA Shredder Mini Spin Column (Qiagen, Hilden, Germany) for 3 min at 16,100 r.c.f. to get rid of debris. Proteins were precipitated using 30% trichloroacetic acid for 20 min on ice to inhibit proteolytic activity.22 Subsequently, samples were centrifuged for 10 min at 16,100 r.c.f., the supernatant was discarded and the protein pellet was washed three times with cold acetone. The pellet was dried and resolved in lysis buffer. The pH of the solution was adjusted to 8 by adding 50 mM NaOH. Protein concentration was determined by Bradford Assay (Coomassie Plus (Bradford) Assay Reagent, Thermo Scientific, Braunschweig, Germany) according to the manufacturer’s instructions. To reach sufficient protein concentrations for 2D-difference gel electrophoresis (DIGE), two clinorotation runs per group were pooled leading to five biological replicates.
Qiashredder mini spin column
Manufactured by Qiagen
Sourced in Germany
The QIAshredder mini spin column is a laboratory equipment designed for mechanical disruption and homogenization of biological samples. It facilitates the efficient lysis and shearing of cells or tissues prior to further processing or analysis.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy mini kit, by Qiagen (3 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Coomassie plus bradford assay reagent, by Thermo Fisher Scientific (1 mentions)
Rat tail collagen type 1, by Corning (1 mentions)
Rosiglitazone, by R&D Systems (1 mentions)
Allprep dna rna mini, by Qiagen (1 mentions)
Celltiter glo 3d cell viability assay reagent, by Promega (1 mentions)
Mirvana mirna isolation kit, by Thermo Fisher Scientific (1 mentions)
Matrigel, by Corning (1 mentions)
Rneasy plus micro kit, by Qiagen (1 mentions)
Agilent rna 6000 pico kit, by Agilent Technologies (1 mentions)
Rankl, by R&D Systems (1 mentions)
Modified porcine trypsin, by Promega (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Rna 6000 nano kit, by Agilent Technologies (1 mentions)
Dneasy plant mini kit, by Qiagen (1 mentions)
Tissuelyser, by Qiagen (1 mentions)
Synergy htx multi mode reader, by Agilent Technologies (1 mentions)
Nitrotyrosine elisa kit, by Abcam (1 mentions)
11 protocols using qiashredder mini spin column
1
Protein Sample Preparation for Proteomic Analysis
2
Bone Marrow Mononuclear Cells Differentiation
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Normal and AML patient BMMCs were purchased from Folio Conversant and ProteoGenex. Recombinant human fibronectin was purchased from Millipore. Rat tail collagen type 1 and matrigel were purchased from Corning Inc. CellTiter‐Glo® 3D cell viability assay reagent was purchased from Promega. Human thrombopoietin, human interleukin‐3 (IL3), human stem cell factor, human granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) and human macrophage colony‐stimulating factor (M‐CSF) were purchased from Life Technologies. Rosiglitazone, human erythropoietin (EPO), granulocyte colony‐stimulating factor (G‐CSF), interleukin‐7 (IL‐7) and receptor activator of nuclear factor kappa‐Β ligand (RANKL) were purchased from R&D systems. Human FLT3 ligand was purchased from TONBO Biosciences. Qubit™ dsDNA HS Assay Kit and MirVana miRNA isolation kit were purchased from Thermo Fisher. Organoid harvest media was purchased from Trevigen. RNeasy Plus Micro Kit, QIAshredder mini spin column and AllPrep DNA/RNA Mini were purchased from Qiagen. Agilent RNA 6000 Pico Kit was ordered from Agilent Technologies. FITC‐anti‐human MPO flow kit was purchased from BioLegend. FITC‐labelled anti‐human CD71, FITC‐labelled anti‐terminal deoxynucleotidyl transferase (TDT), and anti‐human CD110 were purchased from BD Biosciences. TRAP staining kit was purchased from B‐Bridge International, Inc.
3
Protein Extraction and Digestion Protocol
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Protein was extracted from approximately 6 × 106 cells using lysis buffer containing 8 M urea and 400 mM NH4HCO3. Briefly, cells were washed three times with cold PBS, treated with lysis buffer and harvested using cell scrapper. Lysates were concentrated with QIA-shredder mini spin column (Qiagen, Germany) following manufacturer’s instruction. Protein quantifications were performed using BCA Protein Assay Kit (Thermo Fisher Scientific). 20 µg of protein were prepared for disulfide bond reduction by adding 45 mM of dithioerythritol (DTE), and incubated for 30 min at room temperature. Alkylation of cysteines was performed by adding 0.1 M iodocetamide, followed by 30 min incubation at room temperature in the dark. Water was added to a concentration of 1 M urea. 400 ng sequencing grade modified porcine trypsin (Promega, Madison, WI, USA) was added for overnight incubation at 37 °C. Afterwards, samples were purified using C18 spin columns (Pierce, Thermo Scientific, IL, USA) complying manufacturer’s instruction. Resulting supernatants were combined and freeze-drying was performed. Peptide samples were stored at −20 °C prior to LC-MS/MS.
4
Mosquito Tissue Dissection and RNA Extraction
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Mosquitoes were cold-anesthetized and kept on ice until dissections were complete. Individual tissues were removed by forceps or scissors and immediately flash-frozen by placing into nuclease-free tubes in a dry-ice/ethanol bath (−76 °C). The following number of mosquitoes was used for each female library: antenna, 100–220; maxillary palp, 126–816; proboscis, 275–797; rostrum, 110–142; brain, 9–18; foreleg, 125; midleg, 100–125; hindleg, 100–138; ovary, 9–25; abdominal tip, 50. For male libraries, the following number of mosquitoes was dissected for each tissue library: antenna, 75; rostrum, 40–50; brain, 25; foreleg, 100–125; midleg, 100–125; hindleg, 100–125; abdominal tip, 50. Dissected tissue was stored at −80 °C until RNA extraction.
RNA extraction was performed using the Qiagen RNeasy Mini Kit (Qiagen). Tissue was disrupted with an electric tissue grinder loaded with a disposable RNAse free plastic pestle. For legs, abdominal tip and other cuticle-rich tissue, samples were further disrupted by passing tissue through a QIAshredder Mini spin column (Qiagen). RNA quantity and quality were evaluated using an Agilent BioAnalyzer 2100 and the RNA 6000 Nano Kit (Agilent Technologies).
RNA extraction was performed using the Qiagen RNeasy Mini Kit (Qiagen). Tissue was disrupted with an electric tissue grinder loaded with a disposable RNAse free plastic pestle. For legs, abdominal tip and other cuticle-rich tissue, samples were further disrupted by passing tissue through a QIAshredder Mini spin column (Qiagen). RNA quantity and quality were evaluated using an Agilent BioAnalyzer 2100 and the RNA 6000 Nano Kit (Agilent Technologies).
5
Herb and Plant DNA Barcoding
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Forty-seven plants and herb products widely used in Danish and European cuisine were included: 33 plant species (Table S1 ) to build the barcode database and 14 herbal products (Table S2 ) for authenticity evaluation. Samples were seeds, powders, or fresh or dried plant tissue obtained both from a farm in Helsinge, Denmark (Fuglebjerggaard) and a Danish supermarket.
Total genomic DNA was extracted from all samples with the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) (Peter M Hollingsworth et al., 2011) following the manufacturer’s instructions with the following modifications: 100–200 mg of sample was used as a starting material and mixed with 400 µl lysis buffer. TissueLyser (Qiagen) was used for bead treatment in two cycles of 1 min at 30 Hz. The final step of lysis was the addition of 4 µl RNase A stock solution to the sample followed by incubation for 15 min at 65 °C. After lysing and protein precipitation, the samples were centrifuged for 5 min at 20,000×g, and the supernatant was applied to the QIAshredder Mini spin column (Qiagen) and centrifuged for 5 min at 20,000×g. Then, the flow-through fraction excluding any precipitate was mixed with 1.5 volumes washing buffers AW1 and AW2. Finally, the DNA was eluted in two volumes of 50 µl of pre-heated (65 °C) AE buffer (Table S3 ).
Total genomic DNA was extracted from all samples with the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) (Peter M Hollingsworth et al., 2011) following the manufacturer’s instructions with the following modifications: 100–200 mg of sample was used as a starting material and mixed with 400 µl lysis buffer. TissueLyser (Qiagen) was used for bead treatment in two cycles of 1 min at 30 Hz. The final step of lysis was the addition of 4 µl RNase A stock solution to the sample followed by incubation for 15 min at 65 °C. After lysing and protein precipitation, the samples were centrifuged for 5 min at 20,000×g, and the supernatant was applied to the QIAshredder Mini spin column (Qiagen) and centrifuged for 5 min at 20,000×g. Then, the flow-through fraction excluding any precipitate was mixed with 1.5 volumes washing buffers AW1 and AW2. Finally, the DNA was eluted in two volumes of 50 µl of pre-heated (65 °C) AE buffer (
6
Astrocyte RNA Isolation Protocol
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The astrocytes were seeded in a six-well plate and switched to serum-free medium/full medium 24 h before RNA isolation. The medium was removed, and the cells were washed with 1× sterile PBS. RNA was isolated with an RNeasy mini kit (Qiagen, Hilden, Germany; cat.no:74104), using the protocol provided by the manufacturer, and a QIAshredder mini spin column (Qiagen, Hilden, Germany; cat.no:79656).
7
Quantifying 3-Nitrotyrosine Modified Proteins
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The level of 3-nitrotyrosine modified proteins was determined using Nitrotyrosine ELISA Kit (Abcam Cambridge, UK, ab113848) following the manufacturer’s instructions with slight modifications. The amount of 0.1 g of leaf tissue was homogenized in 150 µl of 50 mM Tris–HCl buffer, pH 7.5 with 1 mM PMSF. The extract was centrifuged on QIAshredder Mini Spin Column (Qiagen) at 14,000×g for 10 min at 4 °C and the supernatant was used for further measurements. The assay was performed as an end-point measurement at 450 nm, after termination the reaction by adding 100 ul of 1 N HCl. Results were normalized considering the total protein content in the homogenized samples and expressed as ng 3NT-BSA per µg of protein. The sample protein concentration in the extract was determined by the Bradford’s method109 (link). Spectrophotometrical measurements were performed using the Synergy HTX Multi-Mode Reader (BioTek) in 3 biological replicates.
8
RNA Isolation from Sorted Cells
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For RNA isolation, sorted cells in Qiazol Lysis reagent were resuspended in a total volume of 700 µl and homogenized with QiaShredder Mini Spin columns (Qiagen; 79654). Further RNA extraction was performed according to the Qiagen RNeasy Micro Kit protocol (Qiagen; 74004). RNA concentration was detected by NanoDrop (PeqLab) detection after blank correction.
9
Robust RNA Extraction from Skin Samples
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Total RNAs were isolated from the treatment and control wound skin samples using PureLink RNA mini kits (USA) according to the manufacturer's instructions. RNA was amplified with random primers as following modifications to inhibit the degradation of RNA by abundant skin RNAases. RNA isolation with potential interference of protein were removed by adding the homogenate in lysis buffer 1 ml with 2-mercaptoethanol 10 μl at room temperature for 3 min and then samples were homogenized using QIAshredder Mini Spin Columns (QIAGEN, USA). Supernatant was recovered and RNA was isolated through a RNeasy mini column with one volumes of chilled ethanol (70%) into a RNase-DNase free tube.
10
RNA Extraction from Epithelial Samples
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The epithelial samples were homogenized in RLT buffer (Qiagen, Hilden, Germany) prior to RNA extraction as described previously [20 (link)]. Briefly, the epithelial samples were processed by mixing with disruption beads and 1 ml of RLT buffer in a homogenizer (FastPrep, FP120 Thermo Electron Corporation) for 30 seconds at a setting of 6.5 m/s. After centrifugation of the homogenate at 14462 × g for 10 minutes (min), the supernatants were passed through QIA Shredder Mini Spin Columns (Qiagen). The filtrates were used for RNA extraction.
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