A Basic Guide to 2-D electrophoresis

“Proteomics” is the large-scale screening of the proteins of a cell, organism or biological fluid, a process which requires stringently controlled steps of sample preparation, 2-D electrophoresis, image detection and analysis, spot identification, and database searches. The core technology of proteomics is 2-D electrophoresis. At present, there is no other technique which is capable of simultaneously resolving thousands of proteins in one separation procedure.

The replacement of classical first-dimension carrier ampholyte pH gradients with well-defined immobilized pH gradients has resulted in higher resolution, improved interlaboratory reproducibility, higher protein loading capacity, and an extended basic pH limit for 2-D electrophoresis. With the increased protein capacity, micropreparative 2-D electrophoresis
has accelerated spot identification by mass spectrometry and Edman sequencing. With immobilized gradients stable as high as pH 12, basic proteins can be separated routinely where previously they were lost due to cathodic drift of carrier ampholyte gradients, or suffered from the limited reproducibility of NEPHGE.

The remarkable improvements in 2-D electrophoresis resulting from immobilized pH gradient gels, together with convenient new instruments for IPG-IEF, will make critical contributions to advances in proteome analysis.

This manual clearly describes the actual and technical basis of the current state-ofthe-art 2-D separations using immobilized pH gradients for the first dimension, it provides detailed protocols for new and experienced users,and it includes an extensive bibliography.