Tissue samples were frozen under liquid nitrogen and stored at –80°C until they were analyzed. At the time of analysis, the tissue samples were homogenized by a hand homogenizer with 50 mM potassium phosphate buffer (pH 7.4), 0.25 M sucrose, 1 mM salicylate, 0.3 mM ethylenediaminetetraacetic acid (EDTA), and a complete protease inhibitor cocktail (Roche Applied Science, Indianapolis, USA), and a 15,000 × g supernatant was produced. The total protein concentration of the supernatant was determined with a Coomassie (Bradford) Protein Assay Reagent (Thermo Fisher Scientific Inc., Rockford, Illinois, USA). Enzyme assays were performed at 25°C in 50 mM potassium phosphate buffer (pH 7.8) containing 0.4-mM EDTA, 0.15 mM xanthine, and 500 μM NAD+. XOR activities were determined by the uric acid formation rate and by monitoring the absorbance change at 295 nm. An extinction coefficient of 9.6 mM−1 cm−1 was used for the uric acid. Photometric experiments were performed with a UV-3210 spectrophotometer (Hitachi High-Technologies Co., Ltd., Tokyo) that was equipped with a temperature-control system. High-performance LC (HPLC) was used to separate and detect the accumulated uric acid. The HPLC conditions for separating and detecting uric acid are described above.
Coomassie bradford protein assay reagent
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Coomassie (Bradford) Protein Assay Reagent is a colorimetric assay used to quantify the total protein concentration in a sample. It is based on the change in absorption spectrum of the Coomassie Brilliant Blue G-250 dye upon binding to proteins.
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2 protocols using coomassie bradford protein assay reagent
1
Quantifying Xanthine Oxidoreductase Activity
2
Liposomal Delivery of OVA and CpG-DNA
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Liposomes were prepared according to the standard thin film hydration method.
To a dry, thin membrane of EYPC and TRX (0 or 30 mol%) (total lipids; 1.25 × 10 -5 mol) and MGlu-HPG (lipids/polymer = 7/3, w/w) was added 500 µL of OVA (4 mg/mL) phosphate buffered saline (PBS) (pH 7.4) and the mixture was sonicated for 2 min using a bath-type sonicator. The liposome suspension was further hydrated by freezing and thawing, and was extruded through a polycarbonate membrane with a pore size of 100 nm. The liposome suspension was centrifuged with the speed of 55,000 rpm for 2 h at 4 °C twice to remove free OVA and CpG-DNA. For CpG-DNA inclusion to liposomes, two complexation methods were examined. In the case of Pre-mix, mixed thin membrane was dispersed by mixture of OVA/CpG-DNA (2.5, 5, 7.5 g/mol lipid) in PBS. Lipid concentration and OVA encapsulation were determined by Test-Wako C (Wako Pure Chemical Industries, Ltd) and Coomassie (Bradford) Protein assay reagent (Thermo-Scientific). CpG-DNA amounts in liposomes were determined by Quant-iT Oligreen ssDNA assay as following procedure. Lipid dispersion was mixed with Triton-X 100 (0.2% vol) and Oligreen ssDNA assay reagent in fluorescence microtiter plate. Microplate was excited at 480 nm and fluorescence emission intensity was detected at 520 nm using Microplate Reader (SH-8000 CORONA ELECTRIC Co., Ltd.).
To a dry, thin membrane of EYPC and TRX (0 or 30 mol%) (total lipids; 1.25 × 10 -5 mol) and MGlu-HPG (lipids/polymer = 7/3, w/w) was added 500 µL of OVA (4 mg/mL) phosphate buffered saline (PBS) (pH 7.4) and the mixture was sonicated for 2 min using a bath-type sonicator. The liposome suspension was further hydrated by freezing and thawing, and was extruded through a polycarbonate membrane with a pore size of 100 nm. The liposome suspension was centrifuged with the speed of 55,000 rpm for 2 h at 4 °C twice to remove free OVA and CpG-DNA. For CpG-DNA inclusion to liposomes, two complexation methods were examined. In the case of Pre-mix, mixed thin membrane was dispersed by mixture of OVA/CpG-DNA (2.5, 5, 7.5 g/mol lipid) in PBS. Lipid concentration and OVA encapsulation were determined by Test-Wako C (Wako Pure Chemical Industries, Ltd) and Coomassie (Bradford) Protein assay reagent (Thermo-Scientific). CpG-DNA amounts in liposomes were determined by Quant-iT Oligreen ssDNA assay as following procedure. Lipid dispersion was mixed with Triton-X 100 (0.2% vol) and Oligreen ssDNA assay reagent in fluorescence microtiter plate. Microplate was excited at 480 nm and fluorescence emission intensity was detected at 520 nm using Microplate Reader (SH-8000 CORONA ELECTRIC Co., Ltd.).
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