Protein Chromatography Lab Report

d. You don’t know the molecular weight (MW) of protein X and you are not able to find that information in the scientific literature. The best way to determine the MW of a protein using an SDS-PAGE gel is to use the protein ladder bands to create a Log(MW) vs. Rf graph and calculate the MW from the line of best fit. What is the equation to calculate the Rf of a protein band? Make a table of the Log(MW) and the Rf values for all 5 protein ladder bands. Describe any trends you see in the table

Column Chromatography From: William McEnanly Materials/Equipment: • Plastic syringes with Luer-Lock, 30 mL, without needles (4). • Space Sand (24 mL, or about 2 Tbsp.) ; available at some toy stores. • Scissors.

This experiment was conducted as per the BCHM 310 Laboratory Manual [3]. The first objective of this experiment was to analyze the purity of the invertase fractions collected during experiment 6, and to determine the molecular weight of LDH-H4, LDH-M4 and invertase subunits. This was accomplished using sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS-PAGE). In this procedure, SDS, a negatively charged amphipathic molecule, was used to denature the proteins and to give each protein a similar charge-to-mass ratio [4]. As a result, most oligomeric proteins separated into individual subunits, and each subunit assumed a rod-like shape [4]. The distance travelled by each subunit, along the polyacrylamide gel, was a function of its molecular weight; where proteins with a greater molecular weight moved a smaller distance than proteins with lower weights [5]. Since SDS is not a reducing agent, and no other reducing agent was added, oligomers with disulfide bonds between subunits would have remained intact [4]. However, this was not expected to be problematic for analyzing invertase or LDH isozymes, as these proteins lack disulfide interactions between their subunits [2,6]. In addition, since invertase and LDH are homo-oligomers, each protein’s subunits were expected to migrate the same distance [2,6].

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.

Traditional reagent strip testing for protein uses the principle of the protein error of indicators to produce a visible colorimetric reaction. The protein (primarily albumin) accepts hydrogen ions from the indicator causing change in color. (Susan King Strasinger, 2008)

After purifying the proteins, the students will be able to compare the molecular weights of the samples by running a denatured SDS polyacrylamide gel (Purification & Size Determination of GFP & BFP, EDVOTEK). This will show students which proteins different from one another and how denaturing can affect the proteins. Background:

The testing of various proteins was performed by comparing the molecular weight of proteins using SDS PAGE. The molecular focus in the lab was the testing of proteins, which are macromolecules consisting of amino acid monomers linked through chemical bonds. These proteins have a hierarchy of structure that consists of folding that determines the direct function of each protein.. The molecular weight of these proteins were measured using SDS PAGE. SDS PAGE stands for sodium dodecylsulfate polyacrylamide gel electrophoresis. SDS is an anion detergent that binds with the protein structures and causes them to separate due to the change in bonding charge. SDS and heat are how the proteins are denatured. The process of denaturing a protein is breaking

The role of a victim advocate starts the moment the sexual/abuse hotline had been contact with a victim’s painful story full of trauma. It begins the moment contact has been made with the victim/survivor and it does not stop until the client does not wish to have any further services or support. The role of the advocate is to see the experience through the victim’s eyes and eventually, aiding the victim in creating a detailed path towards recovery and independence from the trauma itself.

ELISA works on the principle of an antigen binding to specific antibody (lock and key), which can be used as a way to identify quantities of proteins in a small sample of fluid. The specific proteins used in an ELISA are estimated quantitatively. The ELISA test is carried out by incubating the serum that contains the antigen of interest with antibody’s within a well, in order for the antibody’s to bind with the specific antigens. The plate is then washed with a mild detergent in order to remove any proteins that have not been bound. The washing of the plates is carried out between every step in order

Paper Chromatography and Spectrophotometry Used to Identify Amino Acids and Concentration of Protein in Solution Introduction: Paper chromatography was used to characterize known amino acids in solution with an accordant Rf value. These Rf values were then used to identify a solution with unknown amino acids and observed Rf values. An Rf value is calculated using the following formula:Distance Traveled by solute (cm)Distance Traveled by solvent (cm), where the distance traveled by solute is measured from the origin line to the center of the solute spot (Lombard, p19). A polar matrix, composed of cellulose, was utilized in tandem with a nonpolar solvent (Ammonia:Ethanol:dH2O) during paper chromatography (Freeman, et al. p113). This diversity

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

Retention time is the time required to elute a solute to a maximum from a column. It indicate how long it takes for a chemical compound to come out of the HPLC column. In HPLC chromatography, the polar solvent will be attracted to a polar solute that bound to the stationary phase. The polar solvent compete with stationary phase for the bound state. Therefore, the higher the polarity of the solute, the more easily it being affected by the solvent.

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

Neufeld is able to determine both relative and quantified information about the concentration of a protein in various locations in a cell. One technique Neufeld uses frequently is the Western Blot, which employs the concept of gel electrophoresis. After choosing the group of cells she wants to examine, Neufeld treats the cells with a solution of sodium dodecyl sulfate (SDS), which causes the cells to lyse and the proteins to denature. Sodium dodecyl sulfate functions by disrupting the non-covalent interactions between molecules. Because proteins typically exhibit hydrogen bonding, hydrophobic interactions, and other intermolecular forces, their structures are greatly compromised. SDS is composed of a hydrophobic hydrocarbon tail and an ionic sulfate group. When SDS encounters a protein, the hydrocarbon tail dissolves the hydrophobic regions of the protein and the ionic sulfate group disrupts non-covalent ionic bonds. Furthermore, SDS causes the protein to hold a negative charge. To make sure the proteins are completely denatured, they are often boiled. Once the proteins are undoubtedly denatured, they are inserted into an acrylamide gel, through which an electrical current is run. The proteins, attracted to the anode due to their negative charge, migrate through small pores in the gel. Because movement through the pores is faster when the protein is small, the various proteins are essentially resolved according to their sizes; smaller

Enzymes are proteins which control biochemical reactions. Liver and potatoes contain an enzyme called catalase that breaks down hydrogen peroxide into the products water and oxygen. We learned that enzymes are specific to substrates in activity 1. The liver, which was the enzyme, only reacted strongly with one substrate, hydrogen peroxide. The liver also had a very weak reaction with carbonic acid.