BCH2011 Lecture Notes - Lecture 18: Protein Purification, Coomassie Brilliant Blue, Ammonium Sulfate
Document Summary
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.
