Anti Cow Serum Lab Report

The goal of this experiment is to see the anti-cow antibody bind to cow serum only, and we expect to see the anti-cow antibody bind to the spot that had the cow serum. The system we used is the serum from Cow, Horse, Goat, Sheep, and Donkey, Chicken. In order to able to detect and analyze proteins based on their ability to bind to a specific antibody, the SDS-PAGE and Western Blot was performed.
SDS Polyacrylamide Gel Electrophoresis (SDS-PAGE) is a very common technique used to separate proteins by molecular weight under the influence of an applied electrical field and then used to prepare for the Western Blot (#1 Lehninger). It uses a polyacrylamide gel as a support medium and sodium dodecyl sulfate (SDS), which is a detergent, to denature

The purpose of stacking gel is to make sure all the proteins start separating at approximately the same time. The pore size is larger so that it is easier for large protein to move in order to catch up with the smaller protein. As heating, SDS denature the proteins, the proteins are loaded onto the wells on the stacking gel. The denatured proteins have a uniform mass to negative charge ratio. Since the current run from negative (top) to positive (bottom), the proteins move toward the bottom. When the electricity is turned on, the proteins and Tris-glycine enter the stacking gel. In stacking gel with pH 6.8, the N-terminal amino group of the proteins and amino acids are protonated at equilibrium, which makes them less negative. The average electrophoretic mobility is very slow. A Gly-chloride ion boundary is formed since glycine moves slower than chloride ion. However, glycine still runs slightly faster than other proteins. As a result, the glycine keeps pushing the protein towards the chloride ion. In other words, the proteins are trapped between glycine and chloride ion. The proteins form a very tight band inside the stacking gel. Once the protein reaches the resolving gel, the pH changes from 6.8 to 8.8 and the pores are smaller. As pH increases, the N-terminal amino groups are deprotonated. Amino acids and proteins are more negatively charged at equilibrium than in stacking gel. As a result,

It is an analytical method where in a protein sample is electrophoresis on an SDS- PAGE and electro transferred on the nitrocellulose membrane. The transferred protein is detected using specific primary enzymes labeled antibody. Antibodies bind to specific sequences of amino acids, known as the epitope. Because amino acid sequences are different from protein to protein, antibodies can recognize specific proteins among a group of many. Therefore, a single protein can be identified in a cell lysate that contains thousands of different proteins and its abundance quantified through western blot