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