Analysis of proteins by a radical-free and highly reducing method of two-dimensional polyacrylamide gel electrophoresis.

Kaltschmidt and Wittmann made significant advances in protein separation techniques by pioneering the two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) method. However, it possessed two serious weaknesses. One was the extremely strong oxidizing power of the polyacrylamide gel, and the other was the excessively high pH of 4.5 of the second-dimensional gel. Consequently, it could not identify all the ribosomal proteins (r-proteins) of Escherichia coli. The radical-free and highly reducing (RFHR) 2-D PAGE overcame the first weakness by prerunning 2-aminoethanethiol HCl, possessing both the properties of a reducing agent and a radical scavenger. The second weakness was addressed by lowering the pH of the second dimension running buffer to 3.6. These improvements eliminated spot pattern noise and achieved the separation of low molecular weight proteins in the sample gel, establishing a new method of 2-D PAGE with high resolution and highly quantitative performance. Using RFHR 2-D PAGE, additionally, we discovered new ribosomal proteins bL35 and bL36, intact bL31, and identified all ribosomal proteins corresponding to the ribosomal genes of the E. coli genome. We also discovered the ribosomal hibernation state in bacteria and the essential hibernation factors. Analysis of total proteins in E. coli cells using the RFHR method revealed 65 spots exhibiting changes in protein content in the stationary phase.