For me, liquid chromatography is both an elegant and powerful method for Biochemists. This week we have been looking at its application in the analysis of complex protein mixtures exemplified by bovine milk. In order to familiarise you with the methodology, I have "spiked" the milk with an intense blue dye called "Brilliant Blue" and the resin (column material is often called this by older scientists!) is Fast Flow Q Sepharose. The dye is not only coloured, but is negatively charged, which ensures it binds efficiently to the positively charged beads of the resin (below, RHS). If a bright blue band appears at the top of your column, you know that your experiment is progressing well. It was really nice to walk round and see that hardly any columns had run dry and that the dye began to accumulate as I expected.
Cell extracts and fluids often comprise many thousands of individual protein species and milk is no exception. The purification of a single protein from such mixtures may seem challenging, but has been successful in many laboratories. However, in our introductory experiment I hope you all observed that some proteins washed straight through the column (theoretically those with a net positive charge, as the beads are positively charged via a quaternary amine, RHS: this is just the same as like poles repelling in bar magnets) and those with a net negative charge remained bound. The bound fraction was eluted (along with the dye) by the application of increasing concentrations of sodium chloride. What kind of column would you use to capture histones?) By refining this process and collecting smaller fractions at a range of salt concentrations we can begin to tease apart the complexity of the mixture. In this way we wont be "wasting pure thoughts on impure proteins" to paraphrase a Nobel Prize-winning Biochemist, Arthur Kornberg!Key words: ion exchange, chromatography, protein purification, organic dyes
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