When cytosolic proteins from mouse liver were separated by a combined method of native isoelectric focusing (IEF) and blue native electrophoresis, spots at pI 6.7/180,000 Da, pI 6.5/100,000 Da and pI 6.4/70,000 Da were detected by Coomassie Brilliant Blue. After separation and detection, the native enzymes were extracted by native electrophoresis and immobilized on the reverse-phase chromatography media ZipTip to produce an enzyme reactor. The hydrolysis activity of 4-methylumebelliferyl acetate by the spots at pI 6.7/180,000 Da and pI 6.4/70,000 Da was 3.0 and 2.4 times, respectively, greater than that with no enzyme. The method can be applied to systematically produce biological reactors after separation and detection of enzymes by the combined method of native IEF and blue native electrophoresis.

We describe a method for detecting phosphoproteins by dot-blot staining with a Phos-tag Aqua fluorescent dye. By using the method, three types of in vitro protein kinase assays for visualizing Tyr-, His-, and Asp-phosphoproteins, respectively, were performed. The staining procedure, which requires less than 2.5 h to complete, is conducted under conditions of neutral pH that are particularly advantageous for detecting labile His- and Asp-phosphoproteins. This method promises rapid and easy detection of phosphoproteins, and should be useful in high-throughput profiling of in vitro kinase activities or in vitro kinase inhibition.

Protein phosphorylation is a key mechanism that regulates cellular physiological functions such as proliferation, migration, cell cycle progression, apoptosis, and differentiation. Aberrations in kinase activity and subsequent dysregulation of protein phosphorylation occur in the process of carcinogenesis and cancer progression and are considered to be therapeutic targets and biomarkers in oncology. Gel electrophoresis has versatile utility in the study of protein phosphorylation. Phosphorylated proteins can be enriched prior to gel electrophoresis, and the proteins separated by gel electrophoresis are visualized by colorimetric methods or western blotting with antibodies, which specifically detect phosphorylation. Phosphorylated proteins migrate differently from non-phosphorylated proteins in gels containing substrates with affinity for phosphorylation. All these methods can be combined in multiple ways, generating unique data from different viewpoints. The identification of separated proteins can be achieved by mass spectrometry, making it possible to integrate protein and genetic data. Peptide array allow the evaluation of kinase activity in heterogeneous samples. As protein phosphorylation and kinase activity are under the regulation of multiple mechanisms and their statuses influence the structures and functions of the other proteins, a multidisciplinary approach is required, and gel electrophoresis will play an important role in the study of protein phosphorylation.