Cells in the exponential growth phase were harvested from BHI broth, washed in Tris-HCl 50 mM (pH 7.5) and used to generate proteins extracts as described by De Angelis et al. (2001) (link). Equivalent amounts of total protein (60 μg for analytical runs or 200 μg for preparative runs for protein identification) were used for each electrophoretic run. The 2-DE was performed essentially as described by Görg et al. (1988) (link) and Hochstrasser et al. (1988) (link) using a Pharmacia 2-D-Electro Focusing (EF) system (GE Healthcare, Milano, Italy). Gels were stained using Brilliant Blue G-Colloidal Concentrate (Sigma) or an MS-compatible silver method. The protein maps were scanned using LabScan on an ImageScanner (GE Healthcare) and were analyzed using ImageMaster 2D Platinum v.6.0 (GE Healthcare). Three gels from three independent experiments were analyzed, and the spot intensities were normalized (De Angelis et al., 2001 (link)), with the spot quantification for each gel calculated as a relative volume (% vol) that corresponded to the volume of each spot divided by the total volume over the entire image. The comparison between different conditions for the amount of the same protein was carried out as the rate of the relative volume of the same spot found in control (untreated cells) and ethanol or other antimicrobials treated cells (Siragusa et al., 2014 (link)).
Brilliant blue g colloidal concentrate
Manufactured by Merck Group
Sourced in United Kingdom, United States
Brilliant blue G-colloidal concentrate is a laboratory reagent. It is a blue dye used in various biological and biochemical applications, such as protein detection and staining.
Automatically generated - may contain errors
Lab products found in correlation
Ampholites, by Bio-Rad (2 mentions)
Ziptip c18 micro columns, by Merck Group (2 mentions)
Tosyl phenylalanyl chloromethyl ketone treated porcine trypsin, by Merck Group (2 mentions)
Immobilized ph gradient, by Bio-Rad (2 mentions)
Ethylene diamine tetraacetic acid (edta), by Merck Group (2 mentions)
Iodoacetamide, by Merck Group (2 mentions)
Urea, by Merck Group (2 mentions)
Acrylamide, by Merck Group (2 mentions)
Thiourea, by Merck Group (2 mentions)
Imagescanner, by GE Healthcare (1 mentions)
Imagemaster 2d platinum v 6, by GE Healthcare (1 mentions)
Chaps, by Merck Group (1 mentions)
Orbitrap fusion lumos tribrid mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Nanolc ultimate 3000 chromatography system, by Thermo Fisher Scientific (1 mentions)
Trypsin, by Promega (1 mentions)
Cathepsin b, by Merck Group (1 mentions)
Alcian blue, by Avantor (1 mentions)
Ecl kit, by Cytiva (1 mentions)
Chlorpromazine, by Merck Group (1 mentions)
Parthenolide, by Merck Group (1 mentions)
7 protocols using brilliant blue g colloidal concentrate
1
Proteomic Analysis of Antimicrobial Responses
2
Thyroid Hormone Metabolite Identification
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3,5-diiodo-L-thyronine (T2) was purchased from Sigma-Aldrich Corp. (St. Louis, MO). All solvents used were of high-performance liquid chromatography-mass spectrometry (LC-MS) grade (Sigma-Aldrich, St. Louis, MO, USA and Carlo Erba, Milan, Italy). Immobilized pH-gradient (IPG) and ampholites were purchased from Bio-Rad Laboratories, Hercules, CA. Acrylamide, other reagents for polyAcrylamide gel preparation, BN-PAGE, and histochemical staining of respiratory complex activity, as well as CHAPS, urea, thiourea, dithioerythriol, EDTA, iodoacetamide, brilliant blue G-colloidal concentrate, and tosyl-phenylalanyl chloromethyl ketone (TPCK)-treated porcine trypsin were from Sigma-Aldrich. ZipTip C18 micro columns were from Millipore, Bedford, MA, USA.
3
Proteomic Profiling of Protein Interactors
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Pull-down elutions were separated by SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and stained with Brilliant Blue G-Colloidal Concentrate (Sigma-Aldrich) according to the manufacturer’s instructions. Gel bands were excised from the whole gel lane and destained, and proteins were in-gel digested with trypsin (sequencing grade, Promega) overnight. The resulting peptide mixtures were analyzed by liquid chromatography (LC)–tandem mass spectrometry using Orbitrap Fusion Lumos Tribrid Mass Spectrometer (Thermo Electron, Bremen, Germany) online with a nanoLC Ultimate 3000 chromatography system (Dionex, Sunnyvale, CA) through an LC EASY-Spray C18 column from Dionex. All raw LC–mass spectrometry files were processed with MaxQuant software (version 1.5.3.8, www.maxquant.org ) and searched against species-specific UniProt protein sequence databases and common contaminants using the Andromeda peptide search engine with a false discovery rate of 0.01 at both peptide and protein levels.
The lists of proteins identified by mass spectrometry were analyzed as follows. First, contaminants and proteins identified by only one peptide were eliminated. Then, only those proteins with at least 10-fold higher peak surface in the experiment samples versus the controls BirA-GFP were considered as positive hits.
The lists of proteins identified by mass spectrometry were analyzed as follows. First, contaminants and proteins identified by only one peptide were eliminated. Then, only those proteins with at least 10-fold higher peak surface in the experiment samples versus the controls BirA-GFP were considered as positive hits.
4
Proteomics Sample Preparation Procedure
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All solvents used were of high performance liquid chromatography-mass spectrometry (LCMS) grade (Sigma-Aldrich, St. Louis, MO, USA and Carlo Erba, Milan, Italy). Immobilized pH-gradient (IPG) and ampholites were purchased from Bio-Rad Laboratories, Hercules, CA. Acrylamide, other reagents for polyAcrylamide gel preparation, CHAPS, urea, thiourea, dithioerythriol, EDTA, iodoacetamide, brilliant blue G-colloidal concentrate, and tosyl-phenylalanyl chloromethyl ketone (TPCK)-treated porcine trypsin were from Sigma-Aldrich. ZipTip C18 micro columns were from Millipore, Bedford, MA, USA.
5
Cathepsin-B Digestion of PGATyr
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The poly(L-glutamic acid-L-tyrosine) 4:1, sodium salt (PGATyr) (MW: 20–50 kDa; Sigma-Aldrich, UK) (2 mg/mL) was incubated with cathepsin-B (from human placenta; Sigma-Aldrich, UK) (0.6 units/mL) in pH 6.4 and pH 7.4 solutions, for 3 days, in an orbital incubator, at 37 °C. 15 μL aliquots were obtained from the incubating mixture every 24 h and were stored at −20 °C until use. Samples were treated with tris-glycine sodium dodecyl sulphate (SDS) sample buffer (Novex by Life Technologies, UK) as recommended by the manufacturer prior to gradient SDS/ polyacrylamide gradient gel electrophoresis (PAGE). After electrophoresis, gels were stained with 0.5% Brilliant Blue G-Colloidal concentrate (Sigma-Aldrich, UK) for 24 h to expose the protein ladder bands, followed by staining with 0.5% Alcian blue (BDH chemicals, UK) for visualising PGATyr. The gel bands were analyzed using ImageJ® for determining the progress of digestion within the course of 3 days.
6
Immunoblotting analysis of PEDF and TSP1
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Sera of tumor-bearing mice were separated by 4–15% SDS-PAGE and transferred onto PVDF membrane. After blocking in TBST (10 ml Tris-base, 100 ml NaCl and 0.1% Tween 20 (pH 7.5)) containing 5% nonfat dry milk, the membranes were exposed to anti-PEDF or anti-TSP1 (Neomarkers, Fremont, CA, USA) and horseradish peroxidase-conjugated secondary Ab, and developed by chemiluminescence (ECL kit; Amersham, Pittsburgh, PA, USA). Protein loading was controlled by staining the gels with Brilliant Blue G—Colloidal Concentrate (Sigma, St. Louis, MO, USA).
7
Reagents for Cell Culture Experiments
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Haloperidol, chlorpromazine, MG-132, pyrrolidine dithiocarbamate (PDTC), aprotinin, phenylmethylsulfonyl fluoride (PMSF), β-mercaptoethanol, brilliant blue G-colloidal concentrate, Brij 35 solution, diethyl pyrocarbonate (DEPC), dithiothreitol (DTT), N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES), leupeptin, lipopolysaccharide (LPS; Escherichia coli, serotype 0127: B8), parthenolide, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO). 2,2,5,7,8-pentamethyl-6-hydroxychroman (PMC) was obtained from Wako Pure Chemical (Osaka, Japan). SP600125 was purchased from Tocris (Ellisville, MO). Recombinant human transforming growth factor-β1 (TGF-β1) was obtained from PeproTech (Rocky Hill, NJ). The murine anti-MMP-9 monoclonal antibody (mAb) was purchased from Neomarkers (Fremont, CA). Anti-mouse or anti-rabbit immunoglobulin G (IgG) linked to horseradish peroxidase antibodies (Abs) and the Western blotting detection system (ECL+) were purchased from Amersham Biosciences (Buckinghamshire, UK). Tissue culture media, supplements, and fetal bovine serum (FBS) were purchased from Invitrogen (Carlsbad, CA). All other reagents used were of the highest grade available.
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