Small-Scale and Large-Scale Procedures

Abstract

There are occasions when a purification reaches a stage where there is so little protein left that it is difficult even to handle the samples without severe losses. Small-scale (or microscale) operations might be defined as those in which the total amount of protein being processed is of the order of a milligram or less, and/or the volume is less than 1 ml (Figure 10.1) . This small amount may be due to the fact that the protein being isolated makes up a very small proportion of the starting material, so even starting with many kilograms, the amount of the desired protein may be only a milligram before accounting for losses during the isolation procedure. Alternatively, it may be that the raw material is not available in any considerable amount, so even an abundant protein in, for example, a small insect, cannot be handled in any great quantity. Two developments over the past decade and more have resulted in small-scale operations becoming commonplace. These are, first, the refinement of high-performance chromatographic equipment, with recent progress in capillary systems that can handle very small volumes. Second, the fact that an end result of only a few micrograms of protein is useful is that it is sufficient for amino acid sequencing. With an appropriate sequence, the gene may be isolated using degenerate oligonucleotides to hybridize with a recombinant library (see Section 9.1).