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].

The homogenates provided were made by homogenizing tissues in a sucrose phosphate buffer in a 1:20 ratio. The protein concentration in bovine cells was measured by diluting the homogenate with a 1:5 ratio; 50 microliters of homogenate and 200 microliters of water. Then 5 known protein concentration samples which were 0.4, 0.8, 1.2, 1.6, 2.0 mg/ml of bovine serum were used to determine absorbance with a spectrophotometer. Two additional samples were made; one was blank and the other was for the specific homogenate sample. Then 3 microliters of bradford assay reagent, which indicates the amount of protein present

With both the stock substrate and varying enzyme solutions prepared, the Spec20 spectrophotometer was used to investigate the enzymatic activity of β-Galactosidase through an absorbance-based assay. Using LoggerPro software on the computer to analyze the absorption data, the Spec20 was calibrated before each run with 0.5 mL of the tested enzyme concentration at an absorbance of 420 nm. Data collection was then started, instantly followed by the addition of 0.5 mL of the stock 2.5 mM substrate solution, topping off the 1-mL cuvettes. Each of the nine varying enzyme concentrations were split between the team and run for a total of 10 minutes. Upon completion, data from each varying enzyme concentration was copied to a single Excel sheet and used

After doing this we diluted the solution using 10 mL of the initial solution and 40 mL of water, then calculated the absorbance using the spectrophotometer. We repeated the process by using the ratio of initial solution to water. The ratios were 20:30, 30:20, 40:10 and 43:7 with the initial solution first then the amount of water second all in measurements of milliliters and placed into a beaker. Each diluted solution was then placed in the spectrophotometer to find the absorbance and wavelength. All data was saved in the program used for this project, however we also wrote down all the data.

The enzyme activity in the mitochondria-free fraction and the control would be expected to be zero since there would be no mitochondria present and the concentrations of DCIP would be relatively high. The enzyme activity in the mitochondria with malanote fraction would be expected to be low since malanote inhibits the SDH activity, stopping the enzyme activity over time. The calculated concentrations of the DCIP in the fractions tubes (See Appendix) supported the expectations. The fractions without the mitochondria had high concentrations in DCIP, while the mitochondria fraction had a low concentration of

The absorbance is measured using a Plate reader and a Standard curve is generated. Also, the different types of pipetting techniques are assessed in this Assay.

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 are crucial for oxidative phosphorylation (Fernandez-Gomez et al., 2005) and succinate dehydrogenase (SDH) is the only enzyme of the Krebs cycle embedded in the inner mitochondrial membrane (Munujos et al., 1993) and is also known as Complex II; its role in the electron transport chain (ETC) is to donate two electrons to the ETC by oxidizing succinate to fumarate. Electrons are transferred from protein complexes in the ETC to the terminal electron acceptor (molecular oxygen, which gets reduced to water), by ubiquinone and cytochrome c, which transfer electrons from Complex I and II to III, and from Complex III to IV, respectively. SDH passes the electrons to flavin adenine dinucleotide (FAD), reducing it to FADH2, then iron sulfur

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

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.

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.