The amount of free hydroxyproline was quantified using tandem mass spectrometry (LC-MS/MS) in wound tissues similar to our recent rat study [20 (link)]. Metabolite extraction was performed with a modified Bligh Dyer method [21 (link)]. Extracted metabolites from the aqueous phase were dried in a CentriVap Concentrator (Labconco, Kansas city, MO, USA) and then stored at -80°C until analysis. Protein pellets were used to normalize extracted metabolite quantity by using a bicinchoninic acid assay (G-Biosciences, St. Louis. MO, USA). A hydroxyproline (Sigma-Aldrich, St. Louis, MO, USA) standard curve was obtained by running hydroxyproline solution at 1 × 10−3, 1 × 10−4, 1 × 10−5, 1 × 10−6, 1 × 10−7, 1 × 10−8, 1 × 10−9 M concentrations. For hydroxyproline detection, hydrophilic interaction liquid chromatography was performed on a Micro200 LC (Eksigent, Redwood, CA, USA) with a Luna NH2 column (3 μ, 100Å, 150mm by 1.0mm, Phenomenex, Torrance, CA, USA). Samples were analyzed on a 5600+ TripleTOF Mass Spectrometer (AB SCIEX, Framingham, MA, USA) and (132.10 → 86.09) m/z transition was used for hydroxyproline detection.
Bicinchoninic acid assay
Manufactured by G Biosciences
Sourced in United States
The Bicinchoninic acid (BCA) assay is a colorimetric detection and quantification method used to measure the total protein concentration in a sample. It is based on the reduction of copper ions (Cu2+) to cuprous ions (Cu+) by proteins in an alkaline medium, and the subsequent colorimetric detection of the cuprous ions by bicinchoninic acid.
Automatically generated - may contain errors
Lab products found in correlation
Tripletof 5600 mass spectrometer, by AB Sciex (1 mentions)
Centrivap concentrator, by Labconco (1 mentions)
Luna nh2 column, by Phenomenex (1 mentions)
Hydroxyproline, by Merck Group (1 mentions)
Chelating sepharose resin, by GE Healthcare (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Luciferase assay, by Promega (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Pgl3 luciferase reporter vector, by Promega (1 mentions)
Celltiter blue assay kit, by Promega (1 mentions)
3 protocols using bicinchoninic acid assay
1
Quantification of Hydroxyproline in Wound Tissue
2
Purification of polyhistidine-tagged tau
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A Ni-NTA column was used. Chelating sepharose resin (GE Healthcare, UK) was charged with 10 mM NiCl2/CH3COONa pH 4.0 and equilibrated with buffer A (50 mM Na2PO4 pH 7.0, 500 mM NaCl, 10 mM imidazole) before addition of the crude extract. The column was re-washed with buffer A, followed by buffer B (50 mM Na2PO4 pH 7.0, 500 mM NaCl, 25 mM imidazole) and the protein eluted with buffer C (50 mM Na2PO4 pH 7.0, 500 mM NaCl, 500 mM imidazole) followed by overnight dialysis in the presence of 25 μg/ml TEV protease, against dialysis buffer (50 mM Tris HCl pH 7.5, 100 mM NaCl) to cleave the polyhistidine-TEV tag. The dialysed tau was re-purified to isolate the TEV protease-cleaved tau. Briefly, the column was equilibrated with buffer A, the cleaved protein collected as the flow through upon elution and ultra-filtered using Vivaspin 20 (5 kDa cut off) where necessary. At each step of the purification process, aliquots were taken for SDS-PAGE and WB analysis. Protein concentration and purity were estimated using the Bicinchoninic acid assay (G-Biosciences, Missouri, USA) and SDS-PAGE respectively.
3
Liposome-Mediated Transfection of Luciferase Reporter in HeLa Cells
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HeLa cells cultured in EMEM (ATCC) supplemented with 10% fetal bovine serum were transferred to a 96 well plate, in quadruplicate, at a density of 20 000 cells per well and incubated for 24 hours at 37 °C in 5% CO2. Liposomes made with a 1 : 1 ratio of DOPE and TZ lipid were added to 200 ng pGL3 Luciferase Reporter Vector (Promega) at N : P ratios of 2.5, 5 and 10, and incubated at 37 °C for 10 minutes before being diluted in 100 μL of non-supplemented EMEM and added to the cells. Following a four-hour incubation at 37 °C, the media was changed, and the cells were incubated for another twenty hours, at which point the cells were lysed with a cell culture lysis reagent at pH 7.8 composed of 25 mM Tris–phosphate buffer, 0.7 g L−1 1,2-diaminocyclohexane, 10% glycerol, 1% Triton X-100, and 1% protease inhibitor cocktail (Millipore). Total protein content was determined with a bicinchoninic acid assay (G-Biosciences) and luciferase protein expression was quantified by a luciferase assay (Promega). Cell viability was assessed using a Cell Titer Blue assay kit (Promega) based on the manufacturer's instructions. In each of the three independent experiments performed, transfection was compared with cells treated with Lipofectamine 3000 (Thermo), following the manufacturer's instructions, and with DNA treated cells.
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