Production of Crude Extract

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

The enzyme lactate dehydrogenase (LDH) catalyzes the last step of anaerobic glycolysis that is important for the normal function of the body. Purification of LDH is essential to understand its structure and function. The purpose of this experiment was to extract and purify LDH enzyme from chicken muscle tissue using a variety of various. Analytical methods such as activity and protein assay were employed to determine the presence and purity of LDH. The cells were initially disrupted and proteins were solubilized. LDH was purified from the ammonium sulfate precipitated protein mixture by affinity chromatography and its activity was studied by

In order to isolate and purify Lactate Dehydrogenase (LDH), we first extracted LDH from bovine muscle tissue. We minced 8 grams of meat and homogenized using blender. The homogenate was centrifuged and the pellet consisting of membranes, organelles, cytoskeletal components and structural fragments was discarded. The crude extract was subjected to 40% (NH4)2SO4 fractionation. The purpose of this is to remove molecules of lesser solubility than LDH like lipids, fats and low soluble proteins. For this, 9.24 g of was slowly added to crude extract while stirring reaching final solution saturation of 40%. The mixture was then centrifuged and the pellet consisting the contaminants was discarded. From table I, the data indicate that the yield for this was 88% with purification factor of 1.5. The total protein in 40% supernatant was 121 mg which decreased from 210 mg present in crude extract while retaining 88% of LDH suggesting some contaminant proteins were removed. Due to some discrepancies in protein data for crude extract and 40% supernatant, we re-assayed these. Even though week 3 data may not be very reliable, the crude extract assay from week 2 and 40% supernatant assay from week 3 gave the most appropriate data with least error.

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

Objective: This lab was performed to help students understand what enzymes do, what they are, and how they are effected by temperature, pH, and concentration. This lab write up only contains information based on the temperature portion of the lab. Introduction: In lab 5; enzyme activity, yeast solution was used as an enzyme to test the rate that oxygen was produced.

A methodology has been developed for making low compressive strength cores that will be used to experimentally examine the Cold Heavy Oil Production with Sand (CHOPS) process occurring in unconsolidated oil reservoirs. The main objective is to experimentally model wormhole (high permeability channel) development during CHOPS and investigate the effect of various flow parameters such as core permeability and porosity, compressive strength, oil/water viscosity contrast, confining pressure and injection rate. Modeling the wormhole development and propagation will enable us be able to developing approximate Inflow Performance Relationship (IPR) curves.

GST- and MBP- tagged proteins were overexpressed at the similar levels, however, protein recovery in a 100.000g supernatant (soluble fraction) was very poor (data not shown). Additionally GST-tagged proteins were irreversibly sticking to the column which made us focus exclusively on the MBP-tag proteins. Temperature variations (160C – 370C), IPTG concentration (0.5mM – 1mM), duration of induction (3h – 18h), as well as the addition of various molecules (arginine, DTT, glycine) to the supernatant, were tested in a number of experiments to optimize the expression and recovery of recombinant proteins (data not shown). In short, protein expression at 160C with an induction with 0.5mMIPTG for 18h was chosen as optimal. Addition of 1M arginine to the homogenate was also shown to be beneficial for protein recovery, especially after freeze/though cycles. In Figure 1A a representative example of one such an experiment is shown.

The enzyme of interest in this experiment was initially extracted from wheat germ via osmotic rupturing of the cellular membrane to release the water-soluble protein.1 This solution was then centrifuged to sediment various cellular debris into a pellet, leaving a solution of various hydrophilic solutes, including acid phosphates.

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

The clarified supernatant was directly purified using a Ni-NTA Spin Column according to instructions supplied by the manufacturer (QIAGEN). Briefly, after washing the column with wash buffer1 (50 mM NaH2PO4 pH 7.5), the supernatant was loaded onto the column, and the column was washed with wash buffer 2 (50 mM NaH2PO4 pH 7.5, 500 mM NaCl, 0.025 Triton X-100 and 20 mM imidazole). The integrase protein was eluted from the column using elution buffer (50 mM NaH2PO4 pH 7.5, 500 mM NaCl, 0.025 Triton X-100 and 400 mM imidazole). The eluted fraction was dialyzed against PBS, and the purity of the integrase protein was assessed by 15% SDS-PAGE and Western Blot. The protein concentration was measured by the Bradford assay, and the purified IntI protein was frozen with 10% glycerol and stored at -20°C until use.

The technique used in this experiment was western blot to determine the protein levels in different cow’s stages; fetal calf serum, newborn calf serum and cow serum. Western blot is technique commonly used to identify proteins by its movement in the gel electrophoresis. Western blot is use to separate protein based on its molecular weight in gel electrophoresis. The proteins separated in the gel, then transferred to a nitrocellulose membrane using an electron current (1). Finally the membrane is incubated with proteins that would stain with antibodies specific for the wanted protein. SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) is used in this experiment to detect the presence of antibodies in different serum samples

1Department of Veterinary Physiology and Biochemistry; Karnataka Veterinary, Animal & Fisheries Sciences University; Veterinary College, Vinobanagar, Shimoga- 577 204, India, 2Division of Biochemistry, 3 & 4Division of Veterinary Biotechnology, Indian Veterinary Research Institute, Izatnagar-243 122, Bareilly, Uttar Pradesh, India

The oil and gas industries involve a high amount of documentation for both transportation as well as extraction and field services. Activities are highly regulated, and subject to oversight from Federal Energy Regulatory Commission (FERC), the Department of Transportation (DoT), the Environmental Protection Agency (EPA), and the Department of the Interior (DoI). That said, the process of regulation is trending toward a more digitized system, with organizations like FERC having their own submission portals that implement a “fill-in-the-blank” platform to complete forms. However, not all forms have yet been converted to digital submission even in agencies with online portals, and other agencies still remain reliant on

Vapor extraction process for the recovery of heavy oil and bitumen was originally proposed by Dr. Butler during 1989-1991. Since its evolution, vapor extraction has emerged as a very effective and promising technique mainly due to its environment friendly nature and cost effectiveness as compared to other recovery techniques. Vapex is basically an in-situ recovery process which injects a vaporized hydrocarbon solvent in the injection well to reduce the viscosity the heavy oil and bitumen and allows it to drain out of the production well through gravity drainage. Mass transfer of vaporized solvent to heavy oil and the gravity drainage are the key parameters which mainly determine the production rate of heavy oil and bitumen with reduced viscosity. This report provides a brief idea about the current heavy oil and bitumen deposits in Canada, a better insight of the Vapex process and the factors which affect the efficiency of the process. Several factors that scale up the performance of the process and important limitations are also indentified that need further research for future growth of the process.

There is need for exploring new frontiers and production of hydrocarbon from the Niger Delta basin for use in the energy sector. Sequel to this, it is pertinent to have adequate knowledge about the geology the basin to discover and exploit new conventional sources. The geology of the Niger Delta basin is summarized into three depositional belts; Akata, Agbada and the Benin formation. The source rock for oil in the basin is from the Akata formation whilst Agbada formation is the main reservoir. Numerous oil fields have been drilled and developed, including Bonga field located in the deep waters of the basin, 120km offshore Gulf of Guinea. The basin also is proven to be one the richest in the world since oil was first discovered sixty years ago.