Lab Report On Protein Chromatography

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Lab Report 4: Protein Purification: Size Exclusion Chromatography Badrun Nessa Rahman Lab Partner: Briana Tolbert Section 55

Objective: The purpose of today’s laboratory is to understand how size exclusion chromatography plays a role in purifying protein which is an important concept to understand as it helps answer the mysteries which lay behind proteins such as the structure or the enzyme kinetics. The fractions which were collected were used to analyze the amount of target protein in each sample.
Principle of Methods:
In today’s laboratory, 40 1.5 ml micro centrifuge tubes which hold the E.Coli lysate samples will be placed in the BioLogic LP chromatography machine. The chromatography machine that we used in the lab contains glass columns with Sephadex
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Elution is the procedure in which the buffer moves and exits through the glass column. The liquid that is collected is called the eluate. There are many types of chromatography, but the one we will be using in the lab specifically is the size exclusion chromatography. Size exclusion chromatography which stands for SEC is chromatography utilizing gel filtration which splits the molecules based on the dimensions of the molecules. Size exclusion chromatography uses a glass column which has beads. These beads are made of pores and Dextran. Different beads are used depending on the size of the protein which needs to be purified. In addition, the glass column has a filter, which prevents the beads from pouring out of the chromatography machine. A buffer keeps the pH constant, which prevents the protein from denaturing. This buffer then seals the pores and the adjacent spaces close to the beads. Eventually the column forms
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