The muscle proteolysis was evaluated by the myofibrillar fragmentation index (MFI) in duplicate as described by Culler et al. [20 (link)]. Each sample (4 g) was homogenized in presence of cold MFI buffer (100 mM KCl, 20 mM potassium phosphate at pH 7, 1 mM MgCl2, and 1 mM NaN3 in distilled deionized water) and centrifuged at 1000× g for 15 min at 2 °C. The pellet was resuspended in cold MFI buffer (40 mL) and centrifuged again. Finally, the pellet was resuspended in 10 mL cold MFI buffer and vortexed until well mixed. Then, the sample was poured through a polyethylene strainer to remove the connective tissue and the tube was rinsed with an additional cold MFI buffer (10 mL). To determinate the protein content in each suspension, the extract (0.25 mL) was mixed with MFI buffer (0.75 mL) and Biuret reagent (4 mL) (Sigma Aldrich, Alcobendas, Madrid, Spain) and after mixing was placed in the dark for 30 min. Bovine serum albumin (BSA) (Sigma Aldrich) was used as standard at concentrations of 0, 2.5, 5, 7.5, and 10 mg/mL. MFI measurement (approximately 0.5 mg protein/mL solution) was measured spectrophotometrically at 540 nm (Thermo Scientific Multiskan GO; Thermo Fisher, Alcobendas, Madrid, Spain) and it was expressed as absorbance of a myofibrillar protein solution multiplied by 200.
Biuret reagent
Manufactured by Merck Group
Sourced in Spain, Sao Tome and Principe, United States
The Biuret reagent is a colorimetric reagent used to detect and quantify the presence of proteins in a solution. It functions by reacting with peptide bonds in proteins, resulting in the formation of a purple-colored complex that can be measured spectrophotometrically.
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Lab products found in correlation
Thermo scientific multiskan go, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Adrenaline, by Merck Group (1 mentions)
Sodium phosphate buffer, by Merck Group (1 mentions)
Acetylcholine iodide, by Merck Group (1 mentions)
Scopolamine methyl nitrate, by Merck Group (1 mentions)
Sodium valproate, by Merck Group (1 mentions)
5 5 dithiobis 2 nitrobenzoic acid dntb, by Merck Group (1 mentions)
Trichloroacetic acid, by Merck Group (1 mentions)
Pilocarpine hydrochloride, by Merck Group (1 mentions)
Thiobarbituric acid, by Merck Group (1 mentions)
Acetic acid, by Merck Group (1 mentions)
Griess reagent, by Merck Group (1 mentions)
Vitamin c, by Merck Group (1 mentions)
Diazepam, by Roche (1 mentions)
Tryptone, by BD (1 mentions)
Ammonium chloride, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Yeast extract, by BD (1 mentions)
Fluorouracil 5 fu, by Merck Group (1 mentions)
6 protocols using biuret reagent
1
Myofibrillar Fragmentation Index Measurement
2
Anticonvulsant Activity of Vitamin C
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Vitamin C, PTZ, scopolamine methyl nitrate, diethyl ether, pilocarpine hydrochloride, sodium valproate, Biuret reagent, acetylcholine iodide, 5′5-dithiobis-(2-nitrobenzoic acid) (DNTB), adrenaline, acetic acid, dichromate, hydrogen peroxide (H2O2), Tris-Hcl, trichloroacetic acid, thiobarbituric acid, sodium phosphate buffer, Griess reagent were purchased from Sigma Chemical Co., St. Louis (United States), while diazepam was purchased from Roche, Neuilly sur-Seine, France. The minimal dose of chemoconvulsant at which 99% of the animals showed a convulsion was determined based on the doses used by other researchers and by a dose-percentage effect curve (Miller and Tainter, 1944 (link); Ahmadiani et al., 2003 (link)). Vitamin C and sodium valproate were dissolved in distilled water. All solutions were prepared freshly in the day of the experiment and were administered intraperitoneally at a volume of 10 ml/kg, except for distilled water and aqueous extract of P. daemia administered per os at the same volume.
3
Biuret Protein Quantification Protocol
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The protein amount was estimated using the Biuret method (Gornall et al., 1949 (link)). The BSA (Carlsbad) was used as standard. Briefly, 3 ml of Biuret reagent (Sigma–Aldrich) and 10 μl of homogenate were added into test tubes. The contents were mixed by inversion and the absorbance was measured at 590 nm after 2 min against blank (3 ml of NaCl 0.9% mixed with 3 ml of Biuret reagent). The weight of protein was plotted against the corresponding absorbance resulting in a standard curve used to determine the protein in unknown samples. The concentration of protein was expressed in mg/ml of protein in the tissue.
4
Biomolecular Protocols for Cell-Based Assays
5
Icariin-Loaded Hydrogel Release Kinetics
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The constructs made of the icariin-loaded hydrogel were submerged in HBSS and left within a shaking incubator (80 rpm) at physiological temperature for 35 days. At predefined intervals, 1 ml of the supernatant was removed and substituted with an identical quantity of fresh HBSS. The cumulative release of icariin was determined by using UV-vis spectroscopy (Specord 40, Analytik Jena, Germany). The absorption intensity at the wavelength (λ) of 276 nm was correlated to the icariin concentration based on a previously constructed calibration curve. The gelatin release was also determined through the Biuret colorimetric test, wherein the Biuret reagent (Sigma, USA) was used for the detection of the peptide-like bonds. In the presence of such bonds, a Cu 2+ ion reacts with nitrogen per amino acid in the peptide sequence in an alkaline solution and is reduced as Cu + , thereby forming a compound with purple color. To do this measurement, 0.1 ml of the sample solution was taken and mixed with 1.1 ml of the Biuret reagent for 10 min at room temperature. 50 µl of the Folin and Ciocalteau's phenol Reagent (FCR) was then included in the solution and left to react for 30 min. The intensity of the generated color (determined by UV-Vis spectroscopy at λ = 758 nm) is associated with the amount of the released gelatin through a previously established calibration curve.
6
Quantifying Protein in Tempeh and Soybeans
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Tempeh as a test sample, soybean as a comparison sample, phosphate buffer pH 8 (Sigma-Aldrich, CAS number : 9011-18-1) as a solvent, biuret reagent (Sigma-Aldrich, CAS number : B3934) as a reagent, aquades as a solvent, and qualitative filter paper (Whatman, Cat No. 1004.110) as a filter.
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