Lab Report 1

Lab Report 1 - Using Affinity Chromatography for the...

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Using Affinity Chromatography for the Purification of Trypsin *Introduction: -Chromatography is a method used to purify molecules in our bodies such as nucleic acids and proteins. This process works by dividing up the molecule into its constituents. Chromatography takes place either on a piece of paper for paper chromatography or in a column. The column is either made of metal or glass and contains particles or beads in it- this is known as the stationary phase. The other phase is the mobile phase which would contain the solvent or mixture to be separated which moves through the stationary phase. The separation of solutes between the stationary and mobile phase is what causes the mixture to divide into its components. -Affinity Chromatography is derived from the remarkable property of proteins to have the ability to attach to certain molecules and then detach forming a noncovalent bond. Affinity chromatography consists of the column being filled with beads or ligands that bind to the static medium present in the column forming a tight covalent bond. This ligand will then from noncovalent bonds with the protein of interest and in our case trypsin. So when the mixture passes through the column, the trypsin from the impure solution will bind to our ligand and the other molecules will wash through into a waste buffer. To obtain our trypsin that is noncovalently bound to the ligand, we remove it using an elution buffer (with a different pH, salt concentration or the addition of a competitive inhibitor) to disturb the bond between the trypsin and the ligand. “Affinity chromatography is most effective when the interaction of the protein and the molecule that is used as the bait is highly specific” (book).
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This note was uploaded on 02/27/2008 for the course BISC 220L taught by Professor Herrera,mcclure during the Spring '07 term at USC.

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Lab Report 1 - Using Affinity Chromatography for the...

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