Pycoerythrin Lab

677 Words3 Pages
The resin used for the ion exchange column for this lab was DEAE-Sephacel resin. This resin is a weak anion exchange resin that allows for the attraction of proteins. The negatively charged proteins become attracted to the resin and the column is washed to remove unwanted molecules such as lipids, nucleic acids and carbohydrates. Once the column is washed, an elution buffer, that is positive in charge, is allowed to flow through the column. The elution buffer attracts the proteins away from the stationary phase and allows them to slowly move and eventually elute from to column. The spectrum profile appears to support the Pseudanabaena phycobiliprotein band from the SDS-PAGE gel overall. The blue fraction appears to correspond with the PC protein…show more content…
Both of these phycobiliproteins were successfully associated with their predicted colors. The fractions were easily identifiable through there colors as expected. The standard phycobiliprotein that exhibited fluorescence on the gel was PE. The fraction that expressed fluorescence was the pink fraction, both as expected. This further aids in the identification of the pink fraction to be PE by expected color and fluorescence. The sizes and relative charges among the phycobiliproteins were compared. Phycoerythrin appeared to be the heaviest phycobiliprotein since it traveled the least amount through the gel compared to the other samples. Phycocyanin travel the second greatest distance making it the second heaviest. Allophycocyanin run the furthest through the gel indicating it to being the lightest. This pattern is supported by the theoretical values and experimentally determined values for MW of each…show more content…
The stationary phase used in this column was DEAE, which has a net positive charge. Since the elution buffer remained constant in its charge, the protein to elute first would be the one with the lower affinity for the column. Having a lower affinity means that the protein was less attracted to the column, and in this case means that the first protein eluted had a lower net negative charge. The Pink fractions eluted first out of the column, thus it is noted that the pink fractions, PE, had a lower negative charge than the blue fractions, PC. The Iron binding protein remains on the DEAE-Sephacel even after a 300 mM NaCL wash due to its very high charge and the resins ability to trap negatively charged molecules. Because of irons 3+ charge these proteins have an enormous affinity for the positively charged resin. It will take a solution of greater pH and of much high concentration then 300 mM to successful elute the iron binding proteins from the column. Basically a solution that has a positive change roughly the same or higher then the ion exchange resin will be necessary to easily elute the iron binding proteins from the top of the
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