Focused Microwave Irradiation of the Brain Preserves In Vivo Protein Phosphorylation: Comparison with Other Methods of Sacrifice and Analysis of Multiple Phosphoproteins

Details

• Personal Author:
  O'Callaghan JP ; Sriram K
• Description:
  At any point in time, net protein phosphorylation represents the contribution of protein kinase and protein phosphatase activities affecting a specific site on a given substrate. Preservation of phosphorylated proteins in neural tissues has traditionally included flash-freezing or fresh tissue processing following tissue isolation. Rapid heat inactivation of protein kinases and phosphatases by focused microwave irradiation sacrifice represents another method to preserve, in vivo, brain protein phosphorylation state. In this study, we compared preservation of the phosphorylation state of a variety of phosphoproteins in the brain following sacrifice of mice by decapitation, decapitation into liquid nitrogen and focused microwave irradiation. We found that microwave irradiation generally provided the highest and most consistent levels of protein phosphorylation, regardless of the substrates examined in striatum and hippocampus. In general, flash-freezing resulted in the least preservation of phospho-state with ERK1/2 and CREB showing almost complete dephosphorylation. When regions of freshly decapitated brains were homogenized and incubated on ice for 30 min, ERK1/2 phosphorylation was completely lost, whereas it was well preserved in microwaved samples left at room temperature for 2 h. Loss of ERK1/2 phosphorylation in the fresh samples could not be attributed to substrate proteolysis. Our results indicate that focused microwave irradiation sacrifice may be required to achieve biologically relevant data for the in vivo protein phosphorylation state of many phosphoproteins. [Description provided by NIOSH]
• Subjects:
  Animals Laboratories Nitrogen Compounds Occupational Health Safety
• Keywords:
  Animal-studies In-vivo-studies Laboratory-animals Nitrogen-compounds Protein-synthesis Proteins
• ISSN:
  0165-0270
• Document Type:
  Text
• Genre:
  Journal Article
• Place as Subject:
  OSHA Region 3 ; West Virginia
• CIO:
  National Institute for Occupational Safety and Health (NIOSH)
• Division:
  HELD - Health Effects Laboratory Division
• Topic:
  Workplace Safety & Health
• Location:
  North America
• Pages in Document:
  159-168
• Volume:
  135
• Issue:
  1
• NIOSHTIC Number:
  nn:20024713
• Citation:
  J Neurosci Methods 2004 May; 135(1-2):159-168
• Contact Point Address:
  HELD/TMBB, Centers for Disease Control and Prevention-NIOSH, Mailstop L-3014, 1095 Willowdale Road, Morgantown, WV 26505, USA
• Federal Fiscal Year:
  2004
• Peer Reviewed:
  True
• Source Full Name:
  Journal of Neuroscience Methods
• Collection(s):
  National Institute for Occupational Safety and Health
• Main Document Checksum:
  urn:sha-512:6359d6d870e9c7c9177cb26032a786949250fa478e2720ddf26e29ebc2e88907193bebf4b5676004dee21cfc12a635e6b9d2c67905db3849729221a8a13db7af
• Download URL:
  https://stacks.cdc.gov/view/cdc/196365/cdc_196365_DS1.pdf
• File Type:
  [PDF - 517.49 KB ]