The gel-based proteomics approach is a valuable technique for studying the 
characteristics of proteins. This technique has diverse applications ranging 
from analysis of a single protein to the study of the total cellular proteins. 
Further, protein quality and to some extent distribution can be first assessed 
by means of one-dimensional gel electrophoresis and then more informatively, for 
comparative analysis, using the two-dimensional gel electrophoresis technique. 
Here, we describe how to take advantage of the availability of fluorescent dyes 
to stain for a selective class of proteins on the same gel for the detection of 
both phospho- and total proteomes. This enables the co-detection of 
phosphoproteins as well as total proteins from the same gel and is accomplished 
by utilizing two different fluorescent stains, the ProQ-Diamond, which stains 
only phosphorylated proteins, and Sypro Ruby, which stains the entire subset of 
proteins. This workflow can be applied to gain insights into the regulatory 
mechanisms induced by signaling molecules such as cyclic nucleotides through the 
quantification and subsequent identification of responsive phospho- and total 
proteins.