How do the different types of chromatography work? (No Audio)
Date Uploaded: 05/03/2020
Chromatography is a term for a variety of techniques in which a mixture of dissolved components is fractionated as it moves through some type of porous matrix. A glass column is filled with beads of an inert matrix. The mixture of proteins to be purified is dissolved in a solution and passed slowly through the column. Proteins that interact with the matrix are slowed down. Those that do not, pass through more quickly. There are three types of chromatography: • Gel Filtration • Ion-exchange • Affinity Gel filtration chromatography separates molecules based on molecular mass. A gel with pores is used to separate large, medium, and small proteins. The large protein emerged first as it remained in the flowing solvent. The medium and small proteins entered the gel and thus their movement was slowed down. The smallest protein was the slowest as it followed the tortuous path of the gel beads. Ion-exchange chromatography separates proteins based on ionic charge. An ion-exchange resin is used to separate a mixture of two proteins, one with a net positive charge, the other with a net negative charge. In this case we are using a resin with positively charged beads. The protein with the net positive charge is repelled by the positive charges on the beads. Thus it flowed quickly through the column. The protein with the net negative charge is attracted to the positive charged beads. Thus it emerges from the column later. Affinity chromatography separates proteins based upon binding characteristics. A sample containing the insulin receptor plus other proteins is purified by running it through a column of beads coated with insulin. The insulin receptor molecules bind to the insulin on the beads and their movement is slowed or stopped. The other proteins do not bind and thus pass quickly through the column. To elute the bound insulin receptor, the column is flushed with a solvent of different ionic composition that causes the receptor to release the insulin. Affinity chromatography is the most specific type of chromatography. Proteins are amde to interact with their ligands: enzymes with their substrates, receptors with their ligands, antibodies with their antigens, etc. Because of the specificity inherent in protein-ligand binding, affinity chromatography can achieve near-total purification of a protein in a single step.
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