Myelin was isolated from 12 weeks old mouse brains as described23 (link). Brains were homogenized in 0.32M saccharose solution and added gently on top of 0.85M saccharose solution in an ultracentrifugation bucket and centrifuged for 30 minutes at 75.000 x g. Interphase, with roughly purified myelin, was removed washed with water, and centrifuged for 15 min at 75,000 x g. Osmotic shock was performed by water incubation for 15 minutes followed by centrifugation for 15 minutes at 12,000 x g. Pellet was resuspended in 0.35M saccharose solution added on top of 0.85m saccharose solution and centrifuged for 30 minutes at 75,000 x g. Purified myelin was washed with water, and centrifuged for 15 minutes at 75,000 x g. The pellet resuspended in PBS, homogenized and stored at -80°C. Protein concentration of myelin was measured with Lowry DC Protein Assay (Bio-Rad).
Lowry dc protein assay
Manufactured by Bio-Rad
Sourced in United States
The Lowry DC protein assay is a colorimetric-based method for determining the total protein concentration in a sample. It is a modification of the original Lowry method, designed to reduce the interference from certain chemicals commonly found in protein samples. The assay involves the reaction of protein with an alkaline copper tartrate solution and the subsequent reduction of the Folin-Ciocalteu reagent, resulting in the development of a blue color that can be measured spectrophotometrically. The intensity of the color is proportional to the protein concentration in the sample.
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Coomassie brilliant blue g 250, by Bio-Rad (2 mentions)
Bodipy iam, by GE Healthcare (2 mentions)
Bodipy iam, by Thermo Fisher Scientific (2 mentions)
Typhoon imager, by GE Healthcare (2 mentions)
Imagequant tl analysis software, by GE Healthcare (2 mentions)
0.45 m pore size filter, by Sarstedt (2 mentions)
Optima le 80k, by Beckman Coulter (2 mentions)
Complete protease inhibitor cocktail, by Merck Group (2 mentions)
Sodium taurocholate, by Merck Group (2 mentions)
Pepsin, by Merck Group (2 mentions)
α amylase, by Merck Group (2 mentions)
Pancreatin, by Merck Group (2 mentions)
Human thrombin, by Merck Group (2 mentions)
Galactose, by Merck Group (2 mentions)
O phthaldialdehyde reagent, by Merck Group (2 mentions)
Trichloroacetic acid, by Merck Group (2 mentions)
Lysozyme, by Merck Group (2 mentions)
Linoleic acid, by Merck Group (2 mentions)
Mucin, by Merck Group (2 mentions)
Bile salt, by Merck Group (2 mentions)
26 protocols using lowry dc protein assay
1
Purification of Myelin from Mouse Brains
2
Protein Extraction and Western Blot Analysis
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Proteins were extracted using RIPA buffer (Thermo Fisher Scientific) supplemented with 1× EDTA-free complete protease inhibitor cocktail (Roche, Basel, Switzerland) and the phosphatase inhibitors sodium fluoride and sodium orthovanadate (Sigma-Aldrich). Protein concentration was determined using Lowry DC protein assay (Bio-Rad, Hercules, CA, USA). Cell lysates (25–30 μg of protein) were resolved on NuPAGETM 4–12% Bis-Tris gels (Thermo Fisher Scientific) and transferred into PVDF membranes. Membranes were blocked for 1 h with 5% non-fat milk or 5% BSA diluted in Tris-buffered saline 0.1% Tween and probed with the indicated primary antibodies (see Supplementary Table 1 ) overnight at 4 °C. Membranes were incubated with secondary antibodies for 1 h and developed with chemiluminescent horseradish peroxidase substrate Immobilon Western (Millipore, Darmstadt, Germany). Quantifications of western blots were performed with Image J (National Institutes of Health, Bethesda, MD, USA).
3
Heat Stress Protein Solubility Assay
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The 2X cell suspension and a 2X compound solution (6 μM compound in experiment buffer with 2% DMSO) were combined in a 1:1 ratio. Then, each sample (3 μM compound or 1% DMSO control) was incubated for 60 minutes at 37 °C with end-over-end rotation. For each cell type, each of the treated cell suspensions were divided into 48 aliquots (12 temperature points, four replicates) that were all subjected to a heat challenge for 3 minutes, each at a different temperature between 44 and 66 °C. After heating, all temperature points for each test condition were repooled to generate individual integrated samples. Precipitated proteins were pelleted by centrifugation at 30,000g for 20 minutes and supernatants constituting the soluble protein fraction were collected. Then the total protein concentration of each soluble fraction was measured by Lowry DC Protein Assay (BioRad). From each soluble fraction, a volume containing a defined amount of total protein was taken for relative quantification using LC-MS/MS based isobaric labeling methodology as described below.
4
Protein and Carbohydrate Analysis of Jatropha Transgenics
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Protein content in Jatropha transgenic lines was analyzed as described by Focks and Benning [47 (link)], using 50 mg of dried endosperm. Protein amounts were measured by the Lowry DC protein assay (Bio-Rad) using γ-globulin as a standard. To analyze carbohydrate content, 50 mg of dried endosperm were homogenized in 200 μl of assay buffer and centrifuged at full speed. The extracted supernatant was used for carbohydrate quantification using a Total Carbohydrate Assay Kit (Sigma-Aldrich). D-glucose was used as a standard for calibration.
5
Isolation of Midgut Brush Border Membranes
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M. sexta midgut tissues from third instar larvae were dissected and stored immediately at −70°C. BBMVs were prepared by the magnesium precipitation method without protease inhibitors [34 (link)] and stored at −70°C until used. The BBMV protein concentrations were determined with the Lowry DC protein assay (Bio-Rad Laboratories) using BSA as a standard.
6
Quantifying S-nitrosation of Erythrocyte Spectrin
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RBC suspensions were prepared by diluting 500 μL of washed RBCs in PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH = 7.4) containing diethylenetriaminepentaacetic acid (1 μM) and incubated with 10−9-10−2 M concentrations of CysNO for 30 min at RT. CysNO was subsequently removed by ultrafiltration using Pall Nanosep 10 kDA MWCO spin columns. RBCs were re-suspended in lysis buffer with protease inhibitors [150 mM NH4Cl, cOmplete ULTRA Tablets (Roche), in H2O]. After protein determination by Lowry (DC Protein Assay, Bio-Rad, München, Germany), a biotin switch assay was carried out to determine the extent of S-nitrosation. The assay was performed according to manufacturer's protocol using a concentration of 0.8 mg/mL protein (S-Nitrosylated Protein Detection Kit, Cayman, Ann Arbor, USA). Samples were loaded onto a 7% Nupage Novex Tris/Acetate precast gel, and a Western blot was performed as described elsewhere (Cortese-Krott et al., 2017 (link)). Membranes were incubated with a primary mouse anti-spectrin antibody (1:1000) from Sigma Aldrich at 4°C overnight, followed by parallel assessment of spectrin and biotin signals on the same nitrocellulose membrane using anti-mouse Cy3-coupled antibodies (Thermo Fisher) and strepdavidin- coupled to the Cy5 fluorophore (Thermo-Fisher).
7
Fluorescent detection of protein thiols
8
Fluorescent detection of protein thiols
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To detect protein thiols, Bodipy-IAM (Bodipy-iodoacetamide; Invitrogen, Eugene, OR), a fluorescently labeled alkylating agent capable of forming covalent adducts with cysteine thiol groups that are not involved in disulfide linkages was utilized. In brief, PCF samples from individual patients (0 and 4 h post-surgery) were diluted 1:10 with de-ionized water prior to determining protein concentration using the Lowry DC protein assay (Bio-Rad). Subsequently, 5 μg of PCF protein was treated with 500 μM Bodipy-IAM for 30 min to assess protein thiol modifications. The reaction was stopped with 5X SDS-PAGE sample buffer (1M Tris-HCl, pH 6.8, 10% SDS, 30% glycerol, 0.05% bromophenol blue) containing 5% β-mercaptoethanol.
Samples were resolved using 12.5% SDS-PAGE gels and imaged in-gel using a Typhoon imager (GE Healthcare Biosciences, Pittsburgh, PA). Following imaging, gels were immediately stained with Coomassie Brilliant Blue G-250 (coomassie blue; Bio-Rad Laboratories, Hercules, CA) for 1 hour, destained overnight, and imaged using the AlphaView SA imager (Protein Simple, Santa Clara, CA) to evaluate protein loading. The Bodipy-IAM fluorescent signal intensity for albumin was quantified using ImageQuantTL analysis software (GE Healthcare Biosciences, Pittsburgh, PA) and the coomassie blue protein stain for albumin was quantified using the AlphaView SA software.
Samples were resolved using 12.5% SDS-PAGE gels and imaged in-gel using a Typhoon imager (GE Healthcare Biosciences, Pittsburgh, PA). Following imaging, gels were immediately stained with Coomassie Brilliant Blue G-250 (coomassie blue; Bio-Rad Laboratories, Hercules, CA) for 1 hour, destained overnight, and imaged using the AlphaView SA imager (Protein Simple, Santa Clara, CA) to evaluate protein loading. The Bodipy-IAM fluorescent signal intensity for albumin was quantified using ImageQuantTL analysis software (GE Healthcare Biosciences, Pittsburgh, PA) and the coomassie blue protein stain for albumin was quantified using the AlphaView SA software.
9
Preparation of Midgut BBMV from P. xylostella Larvae
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P. xylostella midgut tissues from 3rd instar larvae were dissected and stored immediately at −70 °C. BBMV were prepared by the magnesium precipitation method without protease inhibitors as described by Wolfersberger 199348 (link) and stored at −70 °C until used. The BBMV protein concentrations were determined with the Lowry DC protein assay (BioRad, Hercules, CA) using bovine serum albumin as a standard.
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P. xylostella midgut tissues from 3rd instar larvae were dissected and stored immediately at −70 °C. BBMV were prepared by the magnesium precipitation method without protease inhibitors as described by Wolfersberger 199348 (link) and stored at −70 °C until used. The BBMV protein concentrations were determined with the Lowry DC protein assay (BioRad, Hercules, CA) using bovine serum albumin as a standard.
10
Purification of Myelin from Mouse Brains
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