Isolation of Mitochondria

vulgaris plants, via the formation of a standard curve prepared using varying concentrations of bovine serum albumin (BSA) solution. Following absorbance readings of the various BSA solutions, they were plotted against their concentrations providing an indirect measure for determining protein concentrations of the plant samples within the assay tubes, and through further calculations the sample protein concentration. The mean protein concentration for the control group was calculated to be 3.34 ± 1.30 mg/mL, while the mean treated group concentration was 2.01 ± 1.26 mg/mL. These results similarly like the chlorophyll results correlate with the literature articles, as a reduced protein content within the Paraquat treated plants can be expected to some extent (Chia et al., 1981). This reduction in protein concentration is the result of those superoxide anions produced by Paraquat, disrupting the chloroplast membranes and allowing for intracellular components including some proteins to leak out, hence the decrease in protein concentration in comparison to the non-treated plants (Qian et al., 2009). A slight outlier may exist within the treated groups protein concentrations as one of the groups provided a negative value for protein concentration which is not valid, but even after exclusion of that data value, results are still supportive of the expected outcome. Though these results support the claim of Paraquat toxicity causing membrane deterioration and leakiness, protein concentration values are rather more purposeful when used to analyze malondialdehyde (MDA) values on per mg of protein

The genes which encode for the mitochondria’s component proteins are in 2 separate genetic systems in 2 different locations. One of which is the cell nucleus, but the other is inside the organelle itself. There are relatively few genes inside the

Purpose: To determine the concentration of solute in the potato’s cytoplasm by measuring the change

30 g of spinach leaf tissues were put into 50 ml of 0.5M sucrose buffer. The tissues are smashed and then strained with cheesecloth into the centrifuge tube. The product is centrifuge at low speed (200x g) to pellet large cell debris, and the supernatants are saved. The supernatants are centrifuge at high speed (1000x g) to pellet-out the chloroplasts. Chloroplasts are suspended in a 10% propylene glycol solution. The chloroplasts are placed in a boiling water bath for about 5 minutes. Seven labeled 14 ml tubes (1, 2R, 2W, 2G, 2B, 3, 4) containing 6.5 ml of 0.5M NaCl asay buffer. In tube #1 430 uls of boiled chloroplasts suspension was added and to the rest tubes 430 uls of un-boiled chloroplasts. Tube #3 was cover with foil, and tube #4 was

The role of an enzyme is to catalyse reactions within a cell. The enzyme present in a potato (Solanum Tuberosum) is catechol oxidase. In this experiment, the enzyme activity was tested under different temperature and pH conditions. The objective of this experiment was to determine the ideal conditions under which catechol oxidase catalyses reactions. In order to do this, catechol was catalyzed by catechol oxidase into benzoquinone at diverse temperatures and pH values. The enzyme was exposed to its new environment for 5 minutes before the absorbance of the catechol oxidase was measured at 420 nm using a spectrophotometer. The use of a spectrophotometer was crucial for the collection of data in this experiment. When exposed to hot and cold temperatures, some enzymes were found to denature causing the activity to decrease. Similarly, when the pH was too high or low, then the catechol oxidase enzyme experienced a significant decrease in activity. It can be concluded after completing this experiment that the optimal pH for catechol oxidase is 7 and that the prime temperature is 20º C. Due to the fact that the catechol oxidase was only tested under several different temperatures and pH values, it is always possible to get a more precise result by decreasing the increments between the test values. However, our experiment was able to produce accurate results as to the

The crude protein extraction of E. huxleyi cells that were either grown in 23℃ or 18℃, was prepared by grinding the cells in liquid nitrogen until it was fine powder. The protein extract was denatured and solubilized by adding 800 μL of an extraction buffer (30mM Tris-HCl, 7 M urea, 2 M thriourea, and 4% CHAPS), 8 μL of a protease inhibitor cocktail, 16 μL of B-mercaptoethanol, and shaking it for half an hour at room temperature. This process was done in order for no contamination to occur because, this algae’s electrophoretic separation of proteins concentrations is low.   Then the extract was transferred to a 1.5 ml microfuge tube and it was centrifuged for 30 minutes at 15,000 xg at 4 °C. The liquid portion of the extract was transferred

The cell membranes are the utmost essential organelle that surrounds all living cells. Its purpose is to control what goes in and out of the cells and is accountable for the various other properties of the cells as well. The nucleus and other organelles also have membranes that are practically indistinguishable. Membranes are organised in a mosaic arrangement, comprised of carbohydrates, proteins and phospholipids. This can be seen in Figure 1. The objective of this indirect examination is to study the causes of various solvents and conducts on live beetroot cells. The reason why beetroot cells have been selected for this experiment is because they have a big membrane-bound central vacuole, as seen in Figure 2. The red colour anthocyanin, which provides the beetroot its bright colour is located in the vacuole. The cell membrane encloses the whole beetroot cell. The anthocyanin cannot leak out if the membranes stay unharmed. The red colour can escape if the membranes are hassled or broken.

C1.2.2. Optimization of analysis of movies of mitochondrial transport: In terms of acquiring the data for the large scale screen, we need to address important questions in terms of plate design and statistical analysis that build on our previous experience with such problems 18,44-47. In our initial studies described in C1.1.4, we assessed the effect of disrupting microtubules on mitochondrial transport by screening the plate before and after drug application. We did this because of the statistical power associated with paired measurements and because we were unsure how much well-to-well variation would impact the statistical analysis. Overall, we found that this provided an excellent means of measuring changes in mitochondrial transport. Having refined

The success of the macros was evident through their ability to conduct the entirety of the analysis for this research. This in turn, supports the second hypothesis. However, there is still room for optimization, as the macros have limitations. Firstly, they cannot differentiate between a cluster and a single mitochondrion. This is a problem, especially in the soma where there seemed to be large mitochondrial clusters. Therefore, the results from 0-16µm diameter from the soma were not used for statistical

After the treatment with WECU, the cells were harvested, lysed, and the protein concentrations were quantified using a Bio-Rad protein assay as described in a previous study [You et al., 2017]. In a parallel experiment, the mitochondrial and cytosolic fractions were isolated using a mitochondria isolation kit according to the manufacturer’s instructions. For Western blotting, equal amounts of protein samples were electrophoretically transferred onto polyvinylidene difluoride membranes (Schleicher & Schuell, Keene, NH, USA) following electrophoretic separation on sodium-dodecyl sulfate (SDS) gel. After blocking with TBS-T buffer [20 mM Tris (pH 7.4), 150 mM NaCl, 0.1% Tween 20] containing 5% skim milk, the membranes were probed with specific primary antibodies at 4 °C overnight, and then incubated with the appropriate HRP-conjugated secondary antibodies for 2 h at room temperature. The protein bands were detected using an ECL kit as per the manufacturer’s instructions.

To confirm, the P-values of the liver and the kidney is 0.003, this value is less than the null hypothesis of 0.05, as the value is greater than the null hypothesis it rejects the hypothesis depicting that the liver and kidney fractions are not the same. The comparison of the liver cytosol with the liver mitochondria enables us to determine the activity between the cell fractions, the calculated P-value, 0.0012, that disapproves the null hypothesis as 0.0012 ≤ 0.05 indicates that there is no relationship between the cytosol and mitochondrial fractions. Furthermore, the similarities in the Liver mitochondria and the kidney mitochondria activities of 0.109 ≥ 0.05 supports the null hypothesis, accordingly outlines a relationship between the liver and the kidney tissues in the mitochondrial fraction. Lastly, the kidney cytosol and the kidney present low levels of GST activity in the mitochondrial fraction, compared to the cytosolic, this indicates that the metabolic reactivity of the enzyme is more favorable in this specific location of the tissues. (evidence)

Protein purification is a process that can be employed to separate a single protein from a larger starting material which may be anything from an organ to a cell. Isolating a purified protein from a larger fraction enables further analysis such as determination of amino acid sequence, potential biological function, and even evolutionary relationship. (Cuatrecasas 1970) In this experiment, the enzyme lactate dehydrogenase will be purified, this enzyme is found extensively in human cells and catalyzes the conversion of lactate to pyruvate, an essential part in energy production. LDH is a key part of anaerobic energy production especially within glycolysis in which LDH catalyzes the conversion of the reverse reaction, pyruvate to lactate, generating NAD+ from NADH, reproducing the oxidized form of the coenzyme which can be used for oxidative respiration. (Markert 1963) Due to the fact that number of purification steps correlates with the purity of the protein multiple purification techniques will be used to isolate a pure form of LDH. LDH will be isolated from a larger “cytosol” fraction collected from a homogenized rat liver in a previous fractionation exercise. Of the procedures that will be used to isolate and purify proteins from a larger fractionate are a set of techniques collectively known as chromatography. These techniques all have the same premise, in that they consist of a stationary phase, also known as the

Mitochondria are double membraned cell organelle that plays central role in cellular energy provision . These organelles have their own genome which is small in size and are transmitted exclusively through female germ line. The human mitochondrial DNA (mtDNA) is a double-stranded, circular and has 16 569 bp which contains 37 genes coding for two rRNAs, 22 tRNAs and 13 polypeptides which are required in oxidative phosphorylation.

Previously mitochondria were considered to be static and isolated organelles. However, it has now been established that mitochondria form a complex, interconnected, and highly dynamic network. Mitochondria dynamics also involves changes in mitochondrial morphology, number in the cell and movement along the cytoskeleton. Mitochondrial dynamics is said to be tightly regulated by mitochondrial fusion and fission.

Mitochondria are double membrane organelles that are found in the cytosol in Eukaryotic cells. There are many within the cells, and their function is to produce ATP. The organelle originated from the endosymbiosis theory, which means the bacteria engulfed the cell and formed a mutualism relationship. Mitochondria can be used to measure age, health, and stress levels of individuals. Scientist use mitochondria as a measurement tool by using the reporter gene to produce a protein that when newly made appears green, and then when it ages, it appears red (UVA 2014). Furthermore, if mitochondria appear green they are producing a lot of ATP and functioning properly, if they appear red they are oxidized and are not performing correctly. (UVA 2014)