Anion Exchange Chromatography Lab Report

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Discussion/Conclusion The goal of this experiment was to separate two different proteins (Myoglobin, BSA or Cytochrome C) using an ion-exchange chromatography method via a cation (CM) or anion (DEAE) exchanger, followed by a SDS-PAGE gel electrophoresis technique to find the concentration of the sample proteins, with the help of a Bradford assay, which employs a Coomassie dye to bind to proteins. After all techniques were performed, Protein 1 was found to have an absorbance value of 0.451 and a concentration of 0.019 µg/µL. Protein 2 was found to have an absorbance of 0.373 and a concentration of 0.016 µg/µL. Ultimately, using the anion exchanger (DEAE), negatively charged proteins attached onto the immobilized positively charged proteins…show more content…
Knowing this information is helpful because understanding how these charged proteins work and their binding strengths, can have various implications of this to be used in the health field. Our bodies function at a specific pH, but if one was to have an imbalance of certain charged proteins in their tissues, medication could be formulated that would bind an excess of oppositely charged proteins to itself, similar to the ion chromatography method, and then excrete it from the body. Knowing the basic chemistry of opposite charges and how they will be affected, whether it is the strength of their binding or if they are eluted or not, could become very helpful in…show more content…
This was determined after looking at the gel in Figure 2 and comparing the P1 and P2 bands with the molecular weight ladder from Figure 3. P1 is just under 17 g/mol, indicating its Cytochrome C, and P2 is just below the 75 g/mol mark, indicating it to be BSA (which has a molecular weight of about 66 g/mol). The only unusual thing observed here is that the band for P1 in the gel from Figure 2 that was indicated as Cytochrome C looks unusually high for something that would be around 12 g/mol. It looks as though it is almost equivalent to myoglobin. The only reason why it isn’t so, is because when the first set of protein was eluted through the ion chromatography column, the positively charged protein had been able to come out; the only positively charged protein of the three available was Cytochrome C. In the second set, a pH buffer was added that enabled the negatively charged protein (or the BSA) to come off. This method had provided two charged proteins, of which none could be myoglobin because it isn’t charged; it’s about neutral. Perhaps some errors may have occurred in the process that caused the band to look the way it did. Possible errors that may have occurred could have been too much or too little of a certain protein or reagent may have been added that caused an imbalance of molecular weight. Or maybe, the gel was taken out too soon out of the electric field

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