A Neurochemical Approach to Elucidate Metabotropic vs. Ionotropic Glutamate Receptor Activities in Rat Hippocampal Slices
Avital Schurr, Benjamin M. Rigor in BRAIN SLICES in BASIC and CLINICAL RESEARCH, 2020
Isolation of 3H-inositol monophosphates (3H-IPs) is performed by anion exchange liquid chromatography.61 QMA Sep-Pak cartridges (Millipore Corporation, Milford, MA) are prepared by washing in 10 ml of water, 8 ml of 1.0 M triethylammonium hydrogen carbonate (TEAB, available from Fluka BioChemika, Buchs, Switzerland), and then 10 ml of water. After centrifugation of the homogenate, the supernatant containing water-soluble 3H-inositol phosphates is passed over a QMA Sep-Pak cartridge on an Amersham Super Separator-24 apparatus. The column is washed with 10 ml of water, followed by 4 ml of 0.02 M TEAB solution, to remove 3H-inositol precursor. 3H-IPs are eluted into large glass scintillation vials using 4 ml of 0.1 M TEAB. Scintillation cocktail is added to each vial for counting. Tissue pellets are analyzed for total protein using the Biuret method.62 The data are expressed at DPM 3H-IPs/mg of protein or percent of the basal hydrolysis value in each experiment. This procedure results in constant basal hydrolysis and a linear agonist-induced increase in 3H-inositol monophosphate formation over the course of the 60-min hydrolysis incubation in brain slices.63
Assay of Antibiotics in Mammalian Cell Culture
Adorjan Aszalos in Modern Analysis of Antibiotics, 2020
Add 5 ml of solution C to all assay tubes. Incubate at 37°C for 1 hr or heat to 56—60°C for 5 min to dissolve the cell material. Shake for 5—15 min on a reciprocal shaker to complete the dissolution of cells. The intensity of the biuret color produced by the alkaline copper sulfate tartrate (solution C) with the protein is an indication of the amount of protein solution that can be used to react with the Folin-Ciocalteau reagent to produce a reading within the range of the spectrophotometer. Appropriate dilutions of cell solutions showing a pale to deep violet color should be made and further diluted with solution C to a final volume of 5 ml as outlined in the table:
Preparation and Health Benefits of Rice Beverages From Ethnomedicinal Plants: Case Study in North-East of India
Megh R. Goyal, Arijit Nath, Rasul Hafiz Ansar Suleria in Plant-Based Functional Foods and Phytochemicals, 2021
Biuret test is used as a standard method for the qualitative detection of protein in rice beer. Bovine serum albumin is used as a standard in Biuret test [13, 39, 40]. Reports suggest the presence of protein with concentration of 0.97 mgmL−1 in judima [39], 1.05 mgmL−1 in poro apong [40], 9.63-12.42 mgmL−1 in rice beer prepared by tribes in Tripura [13], 3.2 mgmL−1 in boro, 5.7 mgmL−1 in poro, 4.2 mgmL−1 in judima and 6.2 mgmL−1 in jumai [43].
Gastroprotective effect of leaf extract of two varieties grapevine (Vitis vinifera L.) native wild and cultivar grown in North of Tunisia against the oxidative stress induced by ethanol in rats
Published in Biomarkers, 2020
Nabil Saadaoui, Asma Weslati, Taha Barkaoui, Ikram Khemiri, Wafa Gadacha, Abdelaziz Souli, Moncef Mokni, Mounira Harbi, Mossadok Ben-Attia
After the rat stomachs were promptly excised and washed with 0.9% NaCl, the stomachs tissues were ground in Tris-buffered saline (TBS) pH 7.4, homogenized on ice using an ultra-Turrax. Homogenates were centrifuged at 4 °C (9000 g/15 min), and the supernatant was removed and stored at −80 °C until further analysis for determination of protein, malondialdehyde (MDA) levels, catalase (CAT), superoxide dismutase (SOD) and reduced glutathione (GSH). MDA was quantified according to the method described by (Buege and Aust 1978). The protein content was determined by Biuret colorimetric method (Gornall et al.1949). The activity of CAT was measured using the method of Aebi (1984). SOD activity was determined according to the method described by Misra and Fridovich (1972) and the reduced GSH in the gastric tissue was measured by Ellman’s reaction (Moron et al.1979).
Perindopril mitigates LPS-induced cardiopulmonary oxidative and inflammatory damage via inhibition of renin angiotensin system, inflammation and oxidative stress
Published in Immunopharmacology and Immunotoxicology, 2019
Ehab A. M. El-Shoura, Souty M. Z. Sharkawi, Basim A. S. Messiha, Adel G. Bakr, Ramadan A. M. Hemeida
Tissues were homogenized for 10 min in liquid nitrogen using RIPA buffer containing protease and phosphatase inhibitor cocktail. Tissue residue was removed at 4 °C at 10,000×g for 10 min by ultra-centrifugation. Biuret method was used to measure the concentration of total protein [36]. Equal quantities of total proteins (50 mg) from each group were loaded in each lane and separated by electrophoresis of 12.5% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE). Proteins were subsequently transferred to the PVDF membrane using the method of semidry transfer [37]. At room temperature, the membranes were blocked with 5% nonfat dry milk in Tris-buffered saline containing 0.1% Tween 20 (TBST) for 1 h, and then incubated at 4 °C overnight with the primary anti-Akt (1:500) antibodies. Then, washed by TBST three times for 5 min each and the membranes were incubated for 1 h with alkaline phosphatase-conjugated secondary antibody (1:5000). After that, the membrane was washed with TBST four times for 5 min each. Then, the protein bands were detected by BCIP/NBT substrate detection kit. In relation to β-actin, the produced bands were analyzed by using image J® software (NIH, Bethesda, MD).
Antioxidants and radical scavenging role of Leek (Allium kurrat) against aflatoxin-contaminated peanut
Published in Toxin Reviews, 2018
Mahmoud Balbaa, Heba Omran, Nihad Abdel-Monem, Mohamed El-Sayed, Nabila Abdelmeguid
Malondialdehyde (MDA) was determined by the TBA test, whereas TBA reactive substances (TBARS) including lipid hydroperoxides and aldehydes were expressed in terms of MDA equivalents (Ohkawa et al.1979). The concentration of GSH was determined as previously described (Ellman 1959). GPx was assayed in the presence of hydrogen peroxide as a substrate (Paglia and Valentine 1967). Total GST activity (cytosolic and microtonal) and total SOD were determined as previously described (Nishikimi et al.1972, Habig et al.1974). The assay of nitric oxide (NO) is based on a chemical reduction of nitrate into nitrite by vanadium (III) and the formation of a chromophore detected by the acidic Griess reaction (Pai et al.1990). Catalase (CAT) and GGT were essentially determined as previously described (Beers and Sizer 1952, Saw et al.1983). ALT and AST activities were assayed by the formation of chromogenic dinitrophenylhydrazone of pyruvate and oxaloacetate, respectively (Reitman and Frankel 1957). The total proteins were assayed by measuring the purple biuret complex (Doumas et al.1997). Serum albumin, urea, and creatinine levels were determined as previously described (Fawcett and Scatte 1960, Young et al.1975).
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