Abstract
Hyperphosphorylated tau protein is one of the pathological hallmarks of the brains of Alzheimer's disease patient. Phosphorylation status of the protein has been widely studied by Western blot analysis using phosphorylation dependent antibody. In addition, mobility shift of phosphorylated tau can be used as a manifestation of phospho-tau. We set off SDS-PAGE separation of phosphorylated tau using phosphate-binding tag, dinuclear manganese complex of acrylamide-pendant phos-tagTM from NARD Institute Itd. In particular, we have studied the modulation of tau phosphorylation by multiple tau kinases including cycline-dependent protein kinase 5 (CDK5). We optimized SDS-PAGE conditions to resolve a mixture of phospho-tau from bovine brain extract. For example, 7.5% of acrylamide gel with 100 μM of Mn2+-Phos-tag gave much larger mobility shift than 10% of acrylamide gel without Mn2+-Phos-tag. We found that addition of Mn2+-Phos-tag in acrylamide gel caused difficulty of transfer to blotting membrane, resulting in poor sensitivity for Western blot analysis. To rescue the difficulty, we decreased methanol content in acrylamide to 10% and used ImmobilonTM-PSQ (0.2 μm) for a blot membrane to get better transfer. Using Mn2+-Phos-tag SDS-PAGE, CDK5-phosphorylated tau protein was separated to 3 major bands as a result of mobility shift. The present results suggest that the electrophoretic separation using Mn2+-Phos-tag acrylamide was useful to analyze phosphorylation status of tau. Improvement on the blot efficiency must somehow achieve. [J Physiol Sci. 2008;58 Suppl:S152]
