Preserving in vivo protein phosphorylation by focused microwave irradiation sacrifice is critical for analysis of signaling pathways in the brain.
Sriram-K; Benkovic-SA; Miller-DB; O'Callaghan-JP
Program No. 633.1. 2004 Abstract Viewer/Itinerary Planner. Washington, DC: Society for Neuroscience, 2004 Oct; :1
Protein phosphorylation represents the dominant mode of post-translational modification through which physiological processes are mediated. This is achieved through protein kinase catalyzed transfer of phosphate to serine, threonine or tyrosine residues of a given protein substrate. However, due to the rapidly reversible nature of protein phosphorylation, any given phosphorylation event often can be quite transient. Thus, preserving phosphorylation state, particularly in vivo, is critical, since variations in postmortem activities of kinases and phosphatases are likely to affect net protein phosphorylation, unless these enzymes are rapidly inactivated. In this study, we compared various methods of sacrifice and tissue processing for preserving protein phosphorylation in the brain. After sacrifice of mice by decapitation, decapitation into liquid nitrogen or focused microwave irradiation, tissues were homogenized under various buffer conditions. Immunoblot analyses then were performed using phospho-state specific antibodies. We found that focused microwave irradiation sacrifice generally provided the highest and most consistent levels of protein phosphorylation. regardless of the substrates examined. Decapitation in to liquid nitrogen resulted in the least preservation of phospho-state, with ERK1/2 and CREB showing almost complete dephosphorylation. In immunohistochemical studies, perfusion with 4% formaldehyde alone resulted in better detection of phospho-ERK1/2 immunoreactivity, probably due to preservation of the phospho-state that was not apparent in PBS/formaldehyde perfusions with or without phosphatase inhibitors. Our findings suggest that focused microwave irradiation sacrifice may be required to achieve biologically relevant data for the in vivo protein phosphorylation state of many brain phosphoproteins.
Physiological-function; Cell-function; Cellular-function; Cellular-reactions; Laboratory-animals; Animal-studies; Animals; Central-nervous-system; Brain-function
Abstract; Conference/Symposia Proceedings
Program No. 633.1. 2004 Abstract Viewer/Itinerary Planner