Protein Chromatography Lab Report

Enzymes are catalysts that function to speed up reactions; for example, the enzyme sucrose speeds up the hydrolysis of sucrose, which breaks down into glucose and fructose. They speed up reactions but are not consumed by the reaction that is taking place. The most important of the enzyme is the shape as it determines which type of reaction the enzyme speeds up. Enzymes work by passing/lowering and energy barrier and in doing so; they need to bind to substrates via the active. Once they do, the reaction speeds up so much more quickly than it would without the enzyme. Coenzymes and cofactors aid the enzyme when it comes to binding with the substrate. They change the shape of the active site so the substrate can bind properly and perform its function.

The decolorization after taking out the chromatography paper from the ammonia chamber for a prolonged period

The investigation is showing how enzymes work inside a mammal's stomach. Rennin is the enzyme found in young mammals and has more effect

A sample of three female students at the University of Illinois at Urbana-Champaign was involved in this research where cheek cells were extracted from gargled saline solution and placed in a 15-milliliter tube to centrifuged for two minutes at maximum speed to form a pellet at the bottom. These cells will undergo Polymerase Chain Reaction (PCR) amplification technique to generate billions copies of a particular deoxyribonucleic acid (DNA) sequence so we can increase the success rate and clarity on the gel electrophoresis and sanger sequencing. The tube was then emptied to only have the pellet remaining and 500 microliters of Chelex stock was added to the tube to bind metal ions that can interfere with PCR reaction. The tube was then closed loosely, placed in boiling water for ten minutes to lyse the cells, shaken, then centrifuged for a second time for two minutes. Three microliters of clear supernatant from the Chelex solution was added to the primer to help bind polymerase to the gene of interest during annealing in PCR amplification. Primers were used in the solution for our restriction enzyme, Haelll, to cleave our DNA sequence at the GGCC location, between the guanine (G) and cytosine (C).

The results of our experiment were similar to the actual isoelectric point of BSA with the exception of one data record. The calculated pI for BSA was approximately 4.5 after experimentation in comparison to 4.7, the real pI value of BSA. The isoelectric point was calculated after graphing the averages of each buffer solution and taking the x-intercept of the data. The isoelectric point detailed the pH at which the substance had a net charge of zero. The second strip of pH 6.0 with a value of +5mm, was omitted from calculations to improve the accuracy and precision of data. Buffers with a pH greater than the isoelectric move towards the negative end. Most of the buffer solutions with a pH of 4.7 and greater from the data, migrated to the negative

In the Enzyme Lab, we observed and quantified the activity of an enzyme. We determined how the concentration of an enzyme affects the rate at which hydrogen H202 deteriorate. When performing the lab, we took paper cutouts, dipped in our enzymes, yeast, and placed them into a container of the hydrogen peroxide. We performed this activity many times so that we could create an average and just in case one procedure failed we would have other to go off of. Once we took multiple tests we compared each of the results to make sure the dilution was correct.

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

Biotechnology industry, Research, Pharmaceutical industry used purified proteins in large quantities for their purposes. This able genetic engineer to set up some technics to easily extracted from variable source proteins in large amount.

After determining the concentration of the unknown protein sample (_mg/mL), the isolated protein sample will now be separated based on protein size by gel electrophoresis. Gel electrophoresis allows the movement of a molecule with a net charge using an electric field. In addition, the gel serves as a molecular sieve, which reinforces the separation of proteins. Thus, protein molecules that are large in size are usually found at the top of the gel, whereas the protein molecules that are smaller in size are found at the bottom of the gel. This is because the protein molecules that is smaller in size more easily and faster through the pores of the gel where the large ones move more slowly. Consequently, the type of gel used in this experiment is a polyacrylamide gel electrophoresis (PAGE) which is a vertical gel flowing top to bottom for the proteins to move through the positive pole, which the bottom of the gel. The isolated protein sample was then coated with sodium dodecyl sulfate (SDS), which serves as an anionic detergent and purposely coats the proteins with a negative charge. This allows the proteins to move through the gel from the top of the gel (negative pole) to the bottom of the gel (positive pole). Moreover, β-Mercaptoethanol is also added to the isolated protein sample as it serves as a reducing agent by breaking disulfide bridges allowing the proteins to separate easily throughout the gel based on size. Furthermore, upon loading the isolated protein sample to

As the substrate concentration increases, so does the reaction rate. It does not increase steadily, instead it begins by increasing rapidly then gradually decreases. This presents the idea that there would eventually be a maximum rate of production. However, the graph is not large enough to represent it. This graph does not provide evidence that enzymes physically bind to their substrates. If the graph were to represent this hypothesis, it would be a linear graph. This is because each enzyme would only physically bind with two substrates at a time. Therefore, as the number of substrates increases so would the reaction rate in a 1:2 ratio, creating a linear graph. If the hypothesis was wrong and the substrates and enzymes did not physically

Molecules found in nature (termed “natural products”) are a rich source of compounds with biological activities that are useful as drugs. Marine organisms are a particularly good source of natural products. Imagine that you are a marine biologist searching for new natural products for use as anticancer drugs. While diving at a coral reef near Tahiti, you notice an unusual sponge that you have never seen before. You collect a sample, return to your lab and make a whole cell protein extract.

Total serum protein determination is a standard clinical test that is often used for the diagnosis of diseases involving the kidneys, liver or bone marrow and other metabolic or nutritional disorders1. Blood plasma contains approximately 7% proteins by weight and the fluid that remains after the clotting factors are removed from the plasma is the serum2. Albumin and globulin are the most common blood proteins, and the total protein is the combined measure of the two3. The most abundant protein is albumin, which acts as a carrier of many different molecules in the blood and helps maintain blood volume2. The globulin proteins include many enzymes and elements of the immune system such as antibodies and immunoglobulins2. Changes in

The very first purification step involved the 65% ammonium sulfate cut that resulted in a highest protein concentration out of all the purification steps. The protein concentration went up from 3.925mg/ml from the clarified homogenate to 28.11mg/ml, which is resulted of the pelleting out of the LDH and reducing the volume from 114mL to 5.7mL. Furthermore, the 65% pellet cut was able to recovery 46% of the LDH, while purifying it by 6.8 fold. Also, this step was able to remove 65% of the total proteins, while retaining 35% of it. Due to the increased purity and 46% recovery of LDH we can conclude that most of the removed total protein was non-LDH, contaminating protiens. Unfortunately, since this was only the first purification step, the amount of comtaminating proteins is still fairly high as depicted in Figure 14, where the 65% cut pellet contains several dark bands besides the one at 35kDa.

The enzymes that are being used in this lab, whichever catalase it may be, has an optimal or room temperature of 37°C. When the the chosen enzyme catalase is exposed to hydrogen peroxide, it begins to decompose into the water mixed with the oxygen gas in the air. This means that when an enzyme is damaged, it is no longer able to work as a catalyzer in a chemical reaction.

The function of proteins is described by the structure. The three-dimensional structure of protein can be studied by using nuclear magnetic resonance spectroscopy (NMR) or by X-ray crystallography. This structure of protein also helps to understand the role of amino acids. Their role can further be tested by doing mutation in many different genes. Each amino acid sequence code for different proteins. For the purification of proteins, gene cloning is the best and most successful and helpful technique. Now cloning techniques have increased our capability to analyse and understand proteins. Proteins can be expressed and purified in large quantities by using these techniques (Lodge et al., 2007).77%