Male albino rats weighing about (180 – 220g) were housed in polypropylene cages (47 x 34 x 18cm3) with saw dust (renewed after every 48hrs).The animals were acclimatized to standard laboratory conditions (temperature 25 ± 20 C) and maintained on 12h light and 12 h dark cycle for one month before the start of the experiment. The animals were provided with regular rat chow and drinking water ad libitum. The experiment were approved by the institutional animal ethical committee (IAEC) of CPCSEA (Committee
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above. We were able to visualize purity by analyzing the SDS-PAGE gel (Figure 8). We can clearly see in the gel‚ that the Clarified Homogenate is more pure than the 65% cut LDH. We should have been able to visualize that the best purification step was Affinity Chromatography‚ but during the experiment‚ instead of adding the 5x sample buffer‚ we added phosphate buffer.
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separate the cell into parts and study each part and its function as a separate entity. This understanding can be applied to the inner workings of the cell as a whole and visualized by microscopic techniques. With this in mind‚ in this experiment a homogenate‚ a mixture of cellular materials‚ was made from pea
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excised. For each group‚ 6 rats were used for histological examinations and other 6 for biochemical investigations. One kidney was used for mitochondrial separation while the other was homogenized to prepare 10% homogenate to determine adenosine triphosphate (ATP) content. A part of the homogenate was then centrifuged at 14‚000 rpm for 1 h at 4 °C and the supernatant was used to estimate oxidative stress biomarkers. Protein content in the supernatant was determined according to method described by Lowry
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EXTRACTING AND PURIFYING GENOMIC DNA FROM A RAT LIVER FOR ELECTROPHORESIS Deoxyribonucleic acid (DNA) is a molecule that encodes the genetic instructions used in the development and functioning of all known living organisms and many viruses. Along with RNA and proteins‚ DNA is one of the three major macromolecules essential for all known forms of life. Genomic DNA is the DNA that holds the complete set of genetic data for an organism. In humans‚ the genomic DNA spans 46 chromosomes‚ providing a complete
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Experiment IV: Study of Krebs cycle using Mitochondria from Mung Bean Seedlings INTRODUCTION The mitochondria is a very important organelle in the plant cell because it carries out very important cellular reactions in the cell like the Krebs cycle and oxidative phosphorylation which is how the plants produce ATP from the pyruvate produced through glycolysis (Meyer and Millar‚ 2008). Glycolysis produces a net of 2 ATP for the plant which is not enough for the cell to function while the Krebs cycle
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To do this we removed .5 g of agarose and added it in 25 ml of the buffer solution‚ heated it in the microwave at one minute intervals until the solution was clear‚ and then placed the bottled in a 65°C water bath until the temperature was balanced. Once it was cooled‚ we poured the molten gel into the casting tray with
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in 1.0ml‚ Glycine-NaOH buffer (pH 9)‚ assays were carried at 38°C for 30min [Manasi A et al.‚ 2005]. The reaction was terminated by adding of acetic acid (30%) of 200µl [8‚ 9‚ Manasi A et al.‚ 2005 ]. Supernatant (0.5ml) was added to 1M NaOH (0.5ml) and the absorbance of this solution was measured at 410nm [Manasi A et al.‚ 2005]. One protease unit is defined as increases in 1 OD/min. An azocasein assay was carried out using 1% (w/v) solution of the substrate in buffer [Manasi A et al.‚ 2005]
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concentrations of the fractions were determined in the previous lab through protein quantifications of cellular fraction. These fractions were crude homogenate‚ nuclear‚ mitochondrial‚ and lastly cytoplasmic fraction. These concentration values were then used to calculate how many µL are needed for µg of protein. Water and 3X SDS PAGE sample buffer were added into each fraction. In addition‚ MW standard was also prepared. After the apparatus was assembled‚ chambers were filled‚ and the gel completely
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The enzyme tyrosinase was successively extracted by combining a homogenate of a potato and sodium sulfate with ammonium sulfate. Tyrosinase was successfully extracted by taking advantage of solubility properties of certain proteins. A standard curve was generated indicating dopachrome absorbance values through the use of a spectrophotometer and a computer graphing program. A spectrophotometer was used to measure either the amount of light that passed through a solution (transmittance) of the amount
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