Β-Galactosidase Lab Report

Electrons from CI or CII are then transferred through CoQ to CIII (cytochrome c reductase), then to cytochrome c, another electron carrier, and finally to complex IV (cytochrome c oxidase), where electrons are used to reduce O2 to H2O [4]. CI pumps protons out of the mitochondrial matrix with a stoichiometry of 4 protons to 2 electrons [2]. For every electron, the chain through complexes I, III and IV translocates a total of 5 protons from the mitochondrial matrix to the intermembrane space, creating a membrane potential that is used by the CV (ATP synthase) to generate ATP [5].

By analyzing our results, we saw that the more a concentrated solution is diluted, the lower the absorbance becomes. The light in the spectrophotometer has to have something there in order for it to be absorbed. For example, water nearly has an absorbency of 0 because the light can be transmitted right through it. However, when color or dye has been added to a substance the light cannot be transmitted through it and is absorbed. The whole purpose of a spectrophotometer is to calculate how much of the light is absorbed.

The control and mitochondrial-free fractions had high concentrations of DCIP and low enzyme activities compared to the mitochondria fractions. The mitochondria fraction had higher enzyme activity and low concentration of DCIP compared to the inhibited mitochondria with malanote fraction and the fractions without any mitochondria. Introduction: The mitochondrion is an important part of the basic plant and animal cell, serving a function as a main

6. After incubation, expression was checked with Coomassie stained protein gel, 1 ml sample was removed & centrifuged as described in Step 4. cell pellet was freezed at -20°C and samples. Check expression in both the total cell extract soluble and insoluble fractions . If a fraction of the target protein is insoluble, repeat expression at a lower temperature (15 to

Mitochondrion is the power house of the cell. Starting this experiment we were thinking about, what are the effects of SOD in the mitochondria? There are three types of SOD 1, 2 and 3. Would Sod3 increase the mitochondrial since it is located inside the mitochondria’s matrix? It did the opposite the results show that mitochondrial activity was the lowest with Sod3.

Mitochondria of B. oleracea florets were used because they do not contain chloroplasts (which contain SDH too) and both would be present in the pellet after differential centrifugation (Willeford et al., 1989); thus florets were used so that the source of electrons could be related to the activity of mitochondrial SDH, and isolated by mechanical disruption of the plasma membrane to release subcellular compartments, filtered (to remove unfractured cells and extracellular material) and then suspended components were separated from the mitochondria by differential centrifugation. Succinate was reacted with SDH, and its activity was quantified by DCIP and spectrophotometry to establish the rate of SDH enzyme activity. Three fractions (mitochondria-free (MFF), mitochondria (MF) and mitochondria with malonate (MF-M) fractions) and a control were prepared, and assay buffer was added to each of the four samples to provide a favourable environment to maintain the mitochondrial structure and optimal SDH activity (Schneider and Hogeboom, 1951). The rate of SDH activity was indirectly measured by a standard curve of the absorbance against concentration of DCIP (a colorimetric test). The rate of DCIP absorbance correlated with the rate of change in DCIP concentration (also related to the DCIP’s electron acceptance rate) and was presumed to indicate the rate of SDH activity.

The prepared samples were heated with 12.5% (v/v) β-mercaptoethanol for 5 minutes in a boiling water bath to denature the proteins. The SDS-polyacrylamide gel (Biorad, Any kD, Mini-PROTEAN TGX Gel, 10 well, 30 µl) was loaded with heated protein samples of crude extract, flow through, pre-dialysis, post dialysis, and molecular weight standard (Biorad). The gel was run at 24 mA until the tracking dye reached the bottom of the gel. The gel was removed and placed within a stain (0.4 mg/mL Coomassie Blue R250, 40% (v/v) methanol, 5% (v/v) acetic acid) until bands were

The best one was the twitcher mouse model which occurring human krabbe disease that is caused by a mutation in galactocerebrosidase gene. Mutation analysis of the human GALC gene was facilitated by the cloning and sequencing of GALC cDNA (5). Mutation analysis of the human GALC gene was facilitated by the cloning and sequencing of GALC cDNA . This allowed DNA obtained from Krabbe affected people to be sequenced and analysed against the normal GALC gene. To date there have been over forty mutations identified that cause the galactosylceramidase deficiency of Krabbe disease .The most common mutation in the European population is a 30kb deletion which is associated with a C to T transversion at cDNA position 502. The large 30kb deletion affects the production of galactosylceramidase since it removes a significant portion of the enzyme coding region. This results in the cancellation of 5 amino acids and the insertion of 2 amino acids which impacts on the quaternary structure of the

The results of this experiment helped understand the concept of absorbance, transmittance and concentration. Table 1 illustrates the percent transmittance, transmittance, and absorbance of the made solutions. The percent transmittance was found by placing each test tube in the Spec20 and recording its value. The transmittance is how much light is absorbed in the solution, its formula is shown by Equation 1. The absorbance represents the amount of light absorbed by each solution, this was found using Equation 2.

Mitochondrion is considered the energy fuel of the cell. It is the primary site for the ATP production oxidative phosphorylation (OXPHOS) system. The mitochondrial OXPHOS machinery system is mainly composed of five multisubunits complexes (complexes I–V), which are produced by mitochondrial genome. Mitochondrial electron transport chain (ETC) has several essential physiological roles, in which, it is the main source of ATP production in the cell, in addition, its constituent enzyme complexes are a major source of ROS generation. Previous studies tested the effect of A on the mitochondrial bioenergetics function, and have concluded that direct exposure to A leads to significant impairment in the functionality of mitochondrial electron transport

Cell proliferation assay was carried out using CellTiter 96 aqueous one solution cell proliferation assay kit (Promega, USA). Briefly, upon reaching about 75-80% confluency, 5000 cells/well were plated in 96-well microplate in 100 µL media. After seeding for 24 h, the cells were treated with compounds (50 µM) in triplicate. Doxorubicin (10 µM) was used as the positive control. At the end of the sample exposure period (72 h), 20 µL CellTiter 96 aqueous solution was added.

GFP was successfully expressed. The objective of this project was to successfully express, purify, and characterize GFP using transformation, affinity chromatography, SDS-PAGE, Bradford Assay, and the Western Blot techniques in the laboratory. Due to the singular bands in lanes 3, 4, 7, 8, and 9 at 25kDa of the Western Blot, the presence of GFP can be confirmed. While, due to the dark bands in lanes 7, 8, and 9 at 25kDa by themselves in the SDS-PAGE, purity of GFP can be confirmed. In lane 8 of the SDS-PAGE the smears above the dark band of GFP at 25kDa, are like due to overloading the well with proteins.

The % RSD should be less than 2.0%. The relative standard deviation of six replicate measurements of standard solution was found to be 0.26% (limit NMT 2.0%), which indicates that the system is precise to analyze the sample. Placebo solution was prepared in the same manner as standard and sample preparation. No interference of placebo was found. The placebo showed the highest absorbance at 219.8 nm wavelength spectrophotometrically.

Using the yellow tube, which included everything but starch, as the blank, each group zeroed their spectrophotometer. This was done so that any absorbance observed depends only on the amount of starch present, not on any other reagents (buffer, IKI). To zero the spectrophotometer, the wavelength was first set at 580nm, using knob 3 (45). Next, the groups made sure that the light next to “transmittance” was lit, and the chamber to be tightly closed. Having the chamber empty & closed tightly provides reference for the darkest condition possible. Using knob 1, the transmittance was turned until it read 0.0 (45). Before the groups used their blank test tube to zero the spectrophotometer, each needed to wipe the tube with kimwipes to ensure a clean reading. Turning knob 2, each group was then instructed to zero the absorbance, 0.000. Upon removing the blank, each trial was inserted into the chamber (46). The

John Dies at the End John Dies at the End began with a meeting between David Wong and a reporter to discuss the events that took place in David's life following injection with "soy sauce." John, David's best friend, was the first to be exposed to it. He accepted a syringe containing an unknown substance and injected it during a night out. The "soy sauce" caused his senses to elevate to a near superhuman level for a short amount of time and gave him awareness to the otherworldly demons that creep the earth. David thought that his friend's odd behavior was only a reaction to a cocktail of drugs, alcohol, and sleep deprivation.