Experiment 1: Chromatography
Biochemistry
ABSTRACT
For this experiment, Gel Column Chromatography was utilized to separate protein (catalase) from its contaminant Riboflavin – based on their molecular weight and size using the molecular sieving properties of the sephadex-dextran gel. At the end using all the data acquired, a plot was generated giving the relationship of absorbance vs. the concentration.
INTRODUCTION
Chromatography can be described as the process where analytes are separated due to their varying distribution between two phases, a stationary one (stationary phase) and one that moves (mobile phase). Compounds traveling in the mobile phase interact with the stationary phase. Those that are strongly retained by the stationary phase move slowly, while those that interact only weakly move rapidly. Compounds that move rapidly are thereby separated from the compounds that move slowly.
Biochemists who wanted to study a protein had to isolate the protein directly from its biological source and this required large amounts of source material. Today the development of Recombinant DNA methods has made protein production easier. The birth of DNA technology was achieved by the collaboration two scientists; Stanley Cohen and Herbert Boyer. This technology allows biochemists to put the DNA sequence of any protein into bacterial DNA and use bacterial machinery to make the protein of interest. The foreign DNA is digested with restriction enzymes. The plasmid that the DNA is going into (the vector) is digested with the same restriction enzymes. This leaves behind sequences on the vector and the foreign DNA that are complementary “sticky ends”. DNA ligase enzyme joins the foreign DNA and vector creating the recombinant plasmid. Protein of interest is then purified from all other cellular components. Typical purification schemes typically involve cell lysis, treatment with enzymes to degrade cell wall and/or DNA/RNA, treatment with enzymes that stop...