Applications of Protein Purification Essay

The most surprising results of recording my data was my results for the water. Water was negative for every chemical test. Which is what I expected because it does not have any sugar to pass a carbohydrates test, steroids, fats, nor amino acids to pass the remaining tests, although it has so many health benefits for the human body it. Each food item was different with each extraction of macromolecules. The most proteins in the foods that were tested positive were only there, with six being negative. Out of the whole experiment the lowest number was two, when testing for positive

These results shown from this experiment led us to conclude that enzymes work best at certain pH rates. For this particular enzyme, pH 7 worked best. When compared to high levels of pH, the lower levels worked better. The wrong level of pH can denature enzymes; therefore finding the right level is essential. The independent variable was the amount of pH, and the dependent being the rate of oxygen. The results are reliable as they are reinforced by the fact that enzymes typically work best at neutral pH

2. We measured 1 mL of turnip peroxidase (the enzyme) and 3 mL of neutral buffer (pH corresponding to the test tube number i.e. pH 5 in test tube 5) with a syringe and disposed it into tubes 3, 5, 6, 7, 8, and 10

A protein has multiple existing structures, these are the primary, secondary, tertiary and quaternary structures which occur progressively. A protein is essentially a sequence of amino acids which are bonded adjacently, and interact with one another in various ways depending on the R group that the amino acid contains. There are 20 different amino acids which are able to be arranged in any given order, thus giving rise to a potential 2.433x1018 (4.s.f) different combinations, and therefore interactions between the various amino acids.

Proteins are primarily considered to have one primary function to serve its role in an organism, however studies have observed to have multiple functioning proteins known as moonlighting proteins (Khan et al. 2014). Moonlighting proteins along with primary functions, have secondary functions that are not related to the primary function and does not correlate to the primary or other functions (Khan et al. 2014). The multifunctional proteins play essential roles in carrying out biochemical functions which aids in the cell growth but are not caused by gene fusion and multiple RNA splice variants (Amblee et al. 2015). The discovery of moonlighting proteins was first discovered by Piatigorsky and Wistow while observing crystallins (Khan et al. 2014). Crystallins, are structural proteins that are found in the eye lens that exhibit enzymatic activity to make the lens itself (Khan et al. 2014). Crystallin has a primary function to help form the lens of the eye by acting as a structural protein (Amblee et al. 2015). Besides enzymatic activity, crystallin was observed in other mammals to have secondary functions such as metabolic functions which are helpful in prokaryotic (Khan et al 2014). Most moonlighting proteins are characterized as cytosolic enzymes and chaperons, or in other words helping proteins (Amblee et al 2015). The multifunctional proteins or moonlighting proteins can also be identified as receptors, channel proteins and ribosomal proteins (Khan et al. 2014). Due to the

Specificity just means that specific substrates should bind best to specific enzymes. For example the enzyme lactase should bind best to the substrate lactose, rather than lactase binding well to maltose.

In UNIT 7 you learned about the basics structure as well as the basic functions of deoxyribonucleic acid. You learned that deoxyribonucleic acid carries an “instruction manuel” to produce proteins that are responsible for passing traits from your parents to you. As you demonstrated in the previous activity, deoxyribonucleic acid is made of four nucleotides base pairs: adenine, thymine, cytosine, and guanine. They are often abbreviated as A, T, C, and G. The uniqueness of deoxyribonucleic acid is not particularly in just these 4 subunits, but how they are arranged. In this activity you will recreate protein synthesis.

Proteins are important elements in cellular membranes and give the membranes many of their characteristics. In red blood cells, the meshwork of proteins in and around the membrane gives it strength and flexibility, allowing a cell to squeeze through small capillaries without bursting. Other proteins play roles in transporting material in and out of the cell (Lab Manual, Cell Biology). Polyacrylamide gel electrophoresis (PAGE), with all of its different modifications is probably the most widely-utilized procedure in contemporary biochemistry and molecular biology (Mordacq and Ellington (1994)). In this experiment, we will attempt to determine the molecular weights of the major proteins in the plasma membranes of bovine red blood cells (RBCs). The predictions made are if our protein has similar weights as proteins

In the Affinity Chromatography experiment we were purifying our Con A proteins. In general, affinity chromatography is a technique that is used for isolating a protein, in our case Con A from a large amount of other macromolecules. Our protein of interest is captured using a microbead matrix while we let everything else flow through the column. The Sephadex matrix is made of cross-linked glucose or dextran and because our Con A has an affinity for glucose it is able to bind to those beads. In general, we began by equilibrating our column with NaCl, then poured Jack Bean Meal Extract which so happens to contain Con A through our column, the Con A then binded to the Sephadex beads, and finally we eluded with a dextrose solution so that

P1 and P2 centrifuged for three minutes at 1000rpm. Supernatant transferred to Eppendorf tubes, 1ml of each saved and set aside. P1 diluted by a factor of 100 and loaded in a column with 5mL. 5mL undiluted P2 loaded into a separate column. 10mL Buffer A used to wash the column. A 10mL of low-salt buffer loaded into each column, 1-2mL collected into each cuvette. Cuvettes scanned with a spectrophotometer, blanked with low salt buffer. Fraction contained the most protein identified and isolated into an Eppendorf tube and placed on ice. The same procedure followed for medium salt and high salt, the blank correlated with loaded buffer. The beads cleaned with a 10mL resin cleaning buffer.

The Functions of Proteins Introduction Protein accounts for about three-fourths of the dry matter in human tissues other than fat and bone. It is a major structural component of hair, skin, nails, connective tissues, and body organs. It is required for practically every essential function in the body. Proteins are made from the following elements; carbon, hydrogen, oxygen, nitrogen and often sulphur and phosphorus.

Which gas, oxygen or hydrogen, was produced by the reaction? The hydrogen made the stick pop and the oxygen made the glowing end flare up.

We analyzed the picture of the wells for the protein crystallizations of the lysozyme. Protein crystallization is a method used to form the protein crystals. The protein crystal that we analyzed in our wells are of the chicken lysozyme which forms a lattice. The chicken lysozyme is of extreme purity and had a concentration of greater than 5mg/ml in order for it to be formed into a protein crystal. Our protein was placed in different solution and at varying degrees of temperatures to maximize protein crystal growth. The final step after a protein crystal grew was to place it under X-Ray light and test the crystal for

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 goal of this laboratory was to successfully make a gel and to run an SDS-PAGE and determine molecular weight (MW) of an unknown protein sample by comparing it to Log10 of the migration of molecular weight of the standards.