Two pools of plasma samples were obtained by mixing equal proportions of plasma [31 (link)], from the eight diabetic subjects of the EXE group, collected before and after the completion of the training program, and prefractioned using the ProteoMiner™ kit Large-Capacity Kit (Bio-Rad Laboratories, Hercules, CA, USA) according to the manufacturer's protocol. Proteins elution was carried out with urea lysis buffer (8 M urea, 4% CHAPS, 65 mM DTE, and 40 mM Tris base). The protein concentration of each sample was determined according to Bradford [32 (link)]. Two-dimensional electrophoresis (2-DE) was carried out as previously described [33 (link)]. Analytical gels were stained with silver nitrate [34 (link)]. Semipreparative gels for mass spectrometry analysis were stained with Brilliant Blue R-250 (Sigma-Aldrich, Milan, Italy) according to the manufacturer's procedure. Gel images were acquired by the Fluor-S MAX multi-imaging system (Bio-Rad Laboratories Italy, Milan, Italy), and the data were analyzed with the ImageMaster 2D Platinum software. Method for in gel digestion was adapted from Shevchenko et al. [35 (link)] as previously described [36 (link)]. LC-ESI-MS/MS analysis was performed using a Q-TOF microTM mass spectrometer (Micromass, Manchester, UK) as previously described [37 (link)].
Brilliant blue r 250
Manufactured by Merck Group
Sourced in United States
Brilliant Blue R-250 is a laboratory reagent used for the detection and quantification of proteins in various analytical techniques, such as protein electrophoresis and Western blotting. It is a blue dye that binds to the basic and aromatic amino acid residues in proteins, allowing for the visualization and analysis of protein samples.
Automatically generated - may contain errors
Lab products found in correlation
Tween 20, by Merck Group (2 mentions)
Triton x 100, by Merck Group (2 mentions)
Fluor s max, by Bio-Rad (1 mentions)
Immobilon p membrane, by Merck Group (1 mentions)
Temed, by ICN Biomedicals (1 mentions)
Glycine, by Merck Group (1 mentions)
Horseradish peroxidase, by Merck Group (1 mentions)
Novex zymogram developing buffer, by Thermo Fisher Scientific (1 mentions)
Novex zymogram renaturing buffer, by Thermo Fisher Scientific (1 mentions)
Sodium phosphate na3po4, by Merck Group (1 mentions)
Dulbecco s modified eagle medium dmem, by Thermo Fisher Scientific (1 mentions)
Foetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
Matrigel coated transwell, by Corning (1 mentions)
Human serum albumin (hsa), by Merck Group (1 mentions)
Pyronin y, by Merck Group (1 mentions)
Proteinruler 2, by Transgene (1 mentions)
Bl21 de3 chemically competent cell, by Transgene (1 mentions)
Jm109, by Transgene (1 mentions)
Sypro r orange protein gel stain, by Merck Group (1 mentions)
10 protocols using brilliant blue r 250
1
Plasma Proteome Profiling of Diabetic Subjects
2
Mitochondrial Proteomic Analysis by 2DE-MS
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Mitochondria from 3 × 107 cells were extracted as previously described [33 (link)] and suspended in 8 M urea, 4% CHAPS, 65 mM DTE, and 40 mM Tris base and sonicated for 5 s on ice. Following centrifugation at 21000 g, protein concentration was determined by Bradford assay [38 (link)] and 80 μg of total proteins used for 2-dimensional electrophoresis (2DE). Analytical gels were stained with silver nitrate [39 (link)], while semipreparative gels for mass spectrometry analysis were stained with Brilliant Blue R250 (Sigma-Aldrich, USA). Gel images were acquired by Fluor-S MAX multi-imaging system (Bio-Rad Laboratories Italy, Segrate, Italy), and the data were analysed with ImageMaster 2D Platinum software. Gel digestion was carried out according to Shevchenko's protocol [40 (link)] and LC-ESI-MS/MS analysis was performed using a Q-TOF micro™ mass spectrometer (Micromass, UK) as previously described [41 (link)].
3
Fibronectin Immunoassay: Reagents and Antibodies
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The following antibodies were used: monoclonal mouse anti-human fibronectin antibody number 42042, purchased from QED Bioscience Inc., San Diego, California, USA, and goat anti-rabbit immunoglobulin G number A5420, conjugated with horseradish peroxidase, obtained from Sigma-Aldrich, Germany. The following reagents were applied: sodium metaperiodate and hydrazide LC-biotin obtained from Thermo Scientific, USA; standard fibronectin from human plasma, DMSO (dimethyl sulfoxide), sodium dodecyl sulfate, Triton X-100, Brilliant blue R250, glycine, Immobilon P membranes, dithiothreitol, Tween 20 (polyoxyethylene sorbitan monolaurate), and TMB (3.3′,5,5′-tetramethylbenzidine), all supplied by Sigma-Aldrich, Germany; Sephadex G-25 obtained from Pharmacia, Sweden; HEPES (4-(2-hydroxyethyl) piperazine-1-ethanesulfonic acid) supplied by Fluka, Germany; BLOT-QuickBlocker purchased from Millipore, USA; TEMED purchased from ICN Biomedicals, USA; streptavidin-coated sensor chip (SAP) obtained from XanTec Bioanalytics, Germany. All of the remaining, applied reagents were supplied by POCh Gliwice, Poland.
4
MMP-9 Activity Quantification in Plasma
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MMP-9 enzymatic activity was assayed in plasma by both traditional gelatin zymography and a mouse MMP-9 specific enzymatic activity assay (QuickZyme Biosciences; Leiden, The Netherlands) according to manufacturer instructions. For zymography, equal volumes of plasma from 4–6 mice per group were pooled together and denatured in non-reducing 6x Laemmli sample buffer at a ratio of 1:5. Equal volumes equivalent to 3 μl of undiluted plasma samples were then separated on 8% polyacrylamide gels containing 0.05% gelatin under constant voltage. After electrophoresis, gels were removed from cassettes and enzymes were allowed to re-fold in Novex Zymogram Renaturing Buffer (Life Technologies; Grand Island, NY) at room temperature for 90 minutes, replacing buffer every 30 minutes. Gels were then rinsed and incubated at 37°C in Novex Zymogram Developing Buffer (Life Technologies; Thermo Fisher Scientific; Waltham MA) containing 0.02% sodium azide for 90 hours. Following incubation, gels were soaked for 45 minutes in 20% trichloroacetic acid, then stained with Brilliant Blue R250 (Sigma Aldrich; St. Louis, MO) and de-stained until zones of digestion were visible as clear bands against a blue background.
5
Humanised WKS13 mAb Production
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Humanised WKS13 mAb was prepared from immunised mice, with the Fc fragment replaced with human IgG1 Fc [manuscript under revision, see supplementary data]. 2-hydroxypropyl-β-cyclodextrin (HPBCD), L-leucine, Brilliant Blue R-250, phosphate-buffered saline (PBS) and sodium phosphate (Na3PO4) were purchased from Sigma Aldrich (Saint Louis, USA). Dithiothreitol (DTT), prestained protein ladder (PageRuler™ Plus), Dulbecco's Modified Eagle Medium (DMEM) and foetal bovine serum (FBS) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). All reagents were of analytical grade or higher unless otherwise stated.
6
Microglial Migration Assay with PAP-1
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NCM- and GCM-treated microglia, GL261, GL-15 and GBM 18 cells were incubated 3 h at 37 °C in Boyden chamber with or without PAP-1 (50 nM). Cells adhering to the upper side of the membrane were scraped off, whilst migrated cells were stained with brilliant blue R 250 (Sigma-Aldrich) and counted in more than 20 fields with a 20x objective.
7
Invasion Assay for Irradiated Cells
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Twenty-for hours after irradiation, irradiated or control sub-confluent cells were trypsinized, and plated in invasion medium (DMEM supplemented with 100 IU/mL penicillin G and 100 μg/mL streptomycin, 0.1% BSA and 25 mM HEPES, pH 7.4), at a density of 7 × 103 cells/cm2 on poly-lysine or Matrigel-coated transwells (Corning, 8 μm pore size, Corning, NY, USA). Cells were incubated at 37 °C for 4 h (migration) or for 24 h (invasion) with 2 mM hydroxyurea added to both chambers to prevent cell proliferation, then fixed in ice-cold 10% trichloroacetic acid for 10 min in the presence or absence of 5 µM of TRAM-34. Cells adhering to the upper side of the filter were scraped off, whereas cells invaded through the insert were stained with a solution containing 50% isopropanol, 1% formic acid, and 0.5% (w/v) Brilliant blue R 250 (Sigma-Aldrich) and counted on the entire membrane area (at least 20 fields) with a 20× objective.
8
Transwell Invasion Assay for GL261 Cells
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Sub-confluent GL261 cells were trypsinized, and plated in invasion medium (DMEM supplemented with 100 IU/ml penicillin G and 100 μg/ml streptomycin, 0.1% BSA and 25 mM HEPES, pH 7.4), at a density of 7 × 103 cells/cm2 on matrigel-coated transwells (Corning, 8 μm pore size). Cells were treated and incubated for 48 h at 37°C, then fixed in ice-cold 10% trichloroacetic acid for 10 min. Cells adhering to the upper side of the filter were scraped off, whereas cells invaded through the insert were stained with a solution containing 50% isopropanol, 1% formic acid and 0.5% (w/vol) brilliant blue R 250 (Sigma-Aldrich) and counted in at least 20 fields with a 20x objective.
9
Purification and Characterization of Recombinant Proteins
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Bovine serum albumin (BSA), human serum albumin (HSA), Brilliant blue R–250, Pyronin Y, DAPI and SYPRO (R) Orange Protein Gel Stain were purchased from Sigma Chemicals Co. All other chemicals are of analytical reagent grade. JM109, DH10B, BL21(DE3) Chemically Competent Cell, ProteinRuler I and ProteinRuler II, were purchased from TransGen Biotech (Beijing, China). A Pierce Rapid Gold BCA Protein Assay Kit was purchased from Thermo Fisher Scientific. RecR, RuvA, RuvB, RecA and MutL were cloned into an expression vector, and the proteins were purified. Cell cultures were maintained in the DMEM medium supplemented with 10% heat-inactivated fetal calf serum, 1% penicillin/streptomycin storage solution (100 U/ml penicillin, 100 mg/ml streptomycin), at 37°C in a humidified atmosphere containing 5% CO2. All the solutions were prepared with sterile ultrapure water. The (Z)-1,2-dimethyl-4-(pyridin-2-ylmethylene)-1H-imidazol-5(4H)-one (PyMDI) was synthesized according to the synthesis procedure described in [20 (link)].
10
Western Blotting Reagent Protocol
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Tris, glycine, SDS, dimethylsulfoxide (DMSO), acrylamide and bis-acrylamide were purchased from Euromedex. Ponceau S, p-coumaric acid, bromophenol blue, brilliant blue R250, MgSO4, Ca(NO3)2, COSO4, CuCl2, ZnSO4, Na2M0O4, Na2B4O7, FeSO4, VOSO4, vitamins, HEPES, hydrogen peroxide, Tween 20, luminol, Triton X-100, sucrose, acetic acid, dithiothreitol (DTT), RNase A, and propidium iodide (PI) were obtained from Sigma. KCl, H2SO4 and KH2PO4 were from Merck. NaCl was from Fischer scientific. Tryptone peptone and yeast extract were obtained from Difco (Becton Dickinson). Ammonium persulfate (APS), N,N,N’,N’-tetra methyl ethylenediamine (TEMED), bovine serum albumin and Bradford reagents were purchased from BioRad. Propan-2-ol, ethanol, sorbitol, HCl, MgCl2, MnCl2 and (NH4)2SO4 were from Prolabo. Glycerol was obtained from Acros organique.
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