Radiolabeling for two-dimensional gel analysis.

Radiolabeling is a highly sensitive method for protein detection, which is easily performed by the incorporation of radioactive amino acids into proteins. This makes radiolabeling a method of choice for visualizing proteins separated on two-dimensional (2-D) gels. This chapter presents protocols to determine in vivo labeling conditions and to label proteins for the comparison of protein samples by means of 2-D gel electrophoresis.

Two-dimensional gel electrophoresis of total yeast proteins.

Two-dimensional gel electrophoresis (2-DE) offers the opportunity of separating several hundred proteins from a total yeast cellular extract. A detailed description is provided here of the different steps required for the separation and visualization of radiolabeled yeast proteins on high-resolution (24 cm x 20 cm) 2-D gels. Two methods of protein separation are described.

Differential gel exposure, a new methodology for the two-dimensional comparison of protein samples.

We describe a novel methodology for the comparison of protein samples called differential gel exposure (DifExpo). This method is based on the coelectrophoresis on a two-dimensional (2-D) gel of two protein samples. The samples are differentiated from each other by in vivo radiolabelling, using (14)C- and (3)H-isotopes.

Identification in Saccharomyces cerevisiae of a new stable variant of alkyl hydroperoxide reductase 1 (Ahp1) induced by oxidative stress.

Yeasts lacking cytoplasmic superoxide dismutase (Cu,Zn-SOD) activity are permanently subjected to oxidative stress. We used two-dimensional PAGE to examine the proteome pattern of Saccharomyces cerevisiae strains lacking Cu,Zn-SOD. We found a new stable form of alkyl hydroperoxide reductase 1 (Ahp1) with a lower isoelectric point.