Introduction The purpose of this practical is to purify and detect lysozyme from a mixture of components. Lysozyme is a protein found in high concentration in egg whites and is found to be very useful in the pharmaceutical and food industry as it has a high anti-microbial, anti-viral and anti-parasite properties. In hens, it is said to account for 3.5% of the total protein in a hen egg white. In the food and pharmaceutical industry lysozyme is used to inhibit the growth of harmful microorganism and extends the shelf life of pharmaceutical and food products. In the practical carried out a cation exchanger is used. Lysozyme which is positively charged becomes bonded to the negatively charged solid/stationary phase while the negatively charged …show more content…
This assay is a colorimetric assay based on an absorbance acid shift dye referred to as Coomasie Blue. The dye binds to the protein lysozyme if present in the solution. The more protein present the more dye more protein will become bound to it. A color change occurs from Brown to blue to indicate the presence of lysozyme. This was then read at an absorbance of 595nm to determine the concentration of each fraction …show more content…
This method is based an Antibody antigen interaction. The Indirect method involves the use of two antibodies to detect the antigenic protein lysozyme. The lysozyme is added to a 96 well plate to coat the plastic and a primary antibody is then added which recognizes the antigen. The primary antibody used was from a different species to that of the antigen it would capture and it referred to as rabbit-anti-lysozyme antibody. The secondary antibody would the overlap the primary antibody by binding to it. The secondary antibody (anti-rabbit HRP labelled) used is conjugated with an enzyme Horse radish peroxidase which then binds to its substrate tetramethyl-benzidine (TMB) to produce a blue color indicating the presence of lysozyme. SDS-PAGE gel electrophoresis is a process which separates proteins based on molecular weight. The purpose of this method is to separate out the lysozyme by its weight. The weight is known to be 14.6Da. To view the protein separation, the gel was placed into coomassie blue to develop a series of bands to detect the protein by its
Last test, inoculation of phenylalanine agar is used to determine if phenylalanine deaminase oxidizes phenylalanine into phenylpyruvic acid and ammonia. Sixth test, is a Multiple Test Media used to determine the physiological characteristics of unknown #398. First test, Inoculation of Kligler 's Iron agar was used to determine the production of hydrogen sulfide from cysteine and fermentation of glucose and lactose. Last test, inoculation of litmus milk is used to determine the fermentation of lactose, casein, lactalbumin, and
Crystal violet was then added for 60 seconds before being washed off with water. The mordant, Gram’s Iodine, was added for another 60 seconds before getting washed off with water. The heat fixed smear was then washed with 95% alcohol until the wash ran clear, leading to the final step of adding Safranin for 45 seconds before being rinsed with water. The slide was finally blot dyed with bibulous paper before it was placed under a microscope to observe the color and shape of the bacterium. 2.2 Litmus Milk Reaction
The flies in vial 1A will demonstrate AO activity while the flies in vial 1B will show no AO activity. So if the flies from vial 1A turn blue after the assay test, then they have the enzyme for AO activity. If the flies from vial 1B present no color change, then they do not have the enzyme for AO activity. In the second part of the lab, the pattern of inheritance of the aldox gene will be determined, whether it is autosomal or sex-linked. The pattern of inheritance will be autosomal and the genotype of the parents will
The Melibiose (MEL), Arabinose ARA, nitrate reduction, and catalase tests were all positive, and the oxidase test was
After, the pellets were removed, 5 drops of Enzyme Color Reagent went into
Starch solution is then placed into the test tube at a quantity of 5 mL. 5 drops of Lugol’s Iodine solution is added to the test tube. If the color changes, then it is known that starches are present in the solution. Proteins are next tested. In order to do this, 5 mL of gelatin solution is added to the test tube. 10 drops of Biuret’s reagent are added to test for protein.
In a non-reducing sugar 3cm cubed and 10 drops of hydrochloric acid is placed in a test tube for a water bath of 5 minutes to be mixing afterwards. Biurets reagent is added to the protein solution to determine it presence. Testing for
Pat McGurrin October 24, 2015 Period #1 Honors Biology Mr. Dinunzio Murder and Meal Lab Analysis Procedure: 1.) Gather all materials: Safety goggles, 250ml beaker, water, hot-plate, test-tubes, paper bag tear, stomach contents, pipette, Biruet solution, Benedict’s solution, and Iodine solution. 2.) Put on safety glasses.
Introduction The purpose of this lab is to use control variables to help identify different macromolecules. Biological systems are made up of these four major macromolecules: carbohydrates, lipids, proteins and nucleic acids. Carbohydrates are sugar molecules (monosaccharides, disaccharides, and polysaccharides) which make them the most abundant macromolecule on the earth. Lipids (oils and fats, phospholipids and steroids) are insoluble in water and perform many functions such as energy source, essential nutrients, hormones and insulators (Lehman, 1955).
The Another medium used was MAC, it is used to isolate and differentiate gram-negative organisms and it is a pink, dusty rose color. Lastly, the Citrate Slant is a green color and it was used as a differential test to examine enzymes. The media were inoculated at 37°C for 48 hours, then it was observed to determine the
1.1 Abstract The purpose of quantitative analysis of protein using a spectrophotometer is to measure the concentration of proteins in a given sample. The experiment is conducted by laboratory method (Biuret Test) and using spectrophotometer to analyze the absorbance of reactants at 540 nm, hence determining the concentration of the proteins in a given sample. The purpose of stopped enzyme assay to study B-galactosidase is to determine the effect of temperature and concentrations of substrate on enzyme activity.
Uncontrolled Environmental conditions Atmospheric conditions The controlled variable Concentration of amylase was kept under control by measuring the amount of amylase used and also it was made sure the percentage of amylase used was 1%. The Amount of amylase/starch used were kept to 5cm3 at all times. Materials needed Beakers Bunsen burner Test tube Thermometer Stopwatch Test plate Glass rod Starch Amylase solution Water bath Iodine solution. Test tube holder Labels Marker Procedure First 5 test tubes were taken and labeled with numbers from 1 to
Along with being found in plants, they are also present in liver cells, kidney cells, leukocytes and erythrocytes. For the concentration of enzyme experiment, the hypothesis was if the concentration of an enzyme increases, then the enzyme activity will increase as well. The hypothesis was proven to be true, because there are more enzymes to react with substrates. For the enzyme—factors affecting, the hypothesis concluded was if the temperature increases, than the enzyme activity will increase. This however was proven wrong, because enzymes become unstable at higher temperatures.
INTRODUCTION: Arginase is an enzyme- enzymes are biological catalyst which drives a reaction at the speed of life. Arginase is a hydrolase, hydrolases catalyze hydrolysis reactions, this is determined via the E.C number (Nelson and Cox 2008). Arginase has the EC number is 3.5.3.1 (Schomburg 2015). The enzyme ‘commission number’ is the arithmetical classification that is used for enzymes which indicates the chemical reaction they catalyze.
In order to utilize casein, bacteria cells secrete proteolytic exoenzymes (amylases, proteases, pectinases, lipases, xylanases and cellulases) outside of the cell that hydrolyze the protein to amino acids. The amino acids can then be used by cells after crossing the cell membrane via transport proteins [169]. Starch hydrolysis test is used to differentiate bacteria based on their ability to hydrolyze starch with the enzyme α-amylase or oligo-l, 6-glucosidase. These enzymes hydrolyze starch by breaking the glycosidic linkages between the sugar subunits. It aids in the differentiation of species from the genera Corynebacterium, Clostridium, Bacillus, Bacteroides, Fusobacterium and members of Enterococcus [170].