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Lecture 18

BCH2011: Textbook summary - Lecture 18 + 19

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Monash University

LECTURE 18 + 19 Column Chromatography The most powerful methods for fractionating proteins make use of column chromatography, which takes advantage of differences in protein charge, size, binding affinity, and other properties. A porous solid material with appropriate chemical properties (the stationary phase) is held in a column, and a buffered solution (the mobile phase) migrates through it. The protein, dissolved in the same buffered solution that was used to establish the mobile phase, is layered on the top of the column. The protein then percolates through the solid matric as an ever-expanding band within the larger mobile phase. Individual proteins migrate faster or more slowly through the column depending on their properties. In ion-exchange columns, the expansion of the protein band in the mobile phase (the protein solution) is caused both by separation of proteins with different properties and by diffusional spreading. As the length of the column increases, the resolution of two types of protein with different net charges generally improves. However, the rare at which the protein solution can flow through the column usually decreases with column length. And as the length of time spent on the column increases, the resolution can decline as a result of diffusional spreading within each protein band. As the protein-containing solution exits a column, successive portions (fractions) of this effluent are collected in test tubes. Each fraction can be tested for the presence of the protein of interest as well as other properties, such as ionic strength or total protein concentration. All fractions positive for the protein of interest can be combined as the product of this chromatographic step of the protein purification. Ion-Exchange Chromatography: Exploits differences in the sign and magnitude of the net electric charge of proteins at a given pH. The column matrix is a synthetic polymer (resin) containing bound charged groups; those with bound anionic groups are called cation exchanges, and those with bound cationic groups are called anion exchanges. The affinity of each protein for the charged groups on the column is affected by the pH (which determines the ionization start of the molecule) and the concentration of competing free salt ions in the surrounding solution. Separation can be optimized by gradually changing the pH and/or salt concentration of the mobile phase so as to create a pH or salt gradient. Gel Filtration Chromatography (Size-Exclusion Chromatography): Seperates proteins according to size. In this method, large proteins emerge from the column sooner than smaller ones. The solid phase consists of cross-linked polymer beads with engineered pores or cavities of a particular size. Large proteins cannot enter the cavities and so take a shorter path through the column, around the beads. Small proteins enter the cavities and are slowed by their more labyrinthine path through the column. Gel filtration chromatography can also be used to approximate the size of a protein being purified. Dialysis: Dialysis is a procedure that separates proteins from small solutes by taking advantage of the protein’s larger size The partially purified extract is placed in a bag or tube made of a semipermeable membrane. When this is suspended in a muc
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