BCH210H1 Lecture Notes - Lecture 11: Affinity Chromatography, High-Performance Liquid Chromatography, Elution

• School: UTSG
• Department: Biochemical Engineering and Applied Chemistry
• Course: BCH210H1
• Professor: Wright

Document Summary

Characterization of protein structure and function requires a pure sample. Protein samples obtained from tissues or by recombinant dna methods are usually mixtures. Methods are needed to purify proteins for analysis. Separated by protein size (large proteins elute first) Resin consists of porous beads with varying pore size. Separates amino acids or peptides by net charge. Used in amino acid analysis to determine protein composition. Attracts and tightly binds negatively-charged peptides/proteins elute with high salt (nacl) or ph gradients. More hydrophobic compounds interact stronger with the column have longer. Chromatography column separates proteins by selectively binding specific proteins. Resin contains covalently bound substrate or ligand that recognizes one protein in the mixture and binds it with high affinity to the column. Pick the ligand (ni for his tag) Determine the number (percentage) of each of the 20 amino acids. Amino acid hydrolysis heat protein at 100 c in concentrated hydrochloric acid.