Determination of Purity and Yield

Abstract

The dual concepts of protein purity and yield are so basic in protein chemistry that it is easy to forget that both of them are almost impossible to define in absolute terms. A protein is totally pure only when it 1s known to contain only a single species uncontaminated with salts or adventitious water. Samples of such a “pure” protein will yield a single band after electrophoresis on a one- or two-dimensional SDSPAGE gel, will elute from a gel filtration, HPLC, or ion exchange column as a single symmetrical absorbance peak, will yield a single set of mass spectrometric, NMR, or W absorbance spectral signals, and where appropriate, will be free of contaminating enzyme activities. However, many tests are used to assess the purity of a product. Ultimately, they all share one property: Rather than prove that the preparation is absolutely pure, each additional test simply provides another example of the failure to detect any contaminating species that might be present. Since absolute purity can never be established, a simple criterion of purity is used routinely, namely, the inability to detect more than a single band of protein after SDS-PAGE. Since onedimensional SDSPAGE is technically far easier than two-dimensional SDS-PAGE, this technique will be described in detail.