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Phospho-CREB and other phospho-proteins: Improved recovery from brain tissue

Authors
Journal
Journal of Neuroscience Methods
0165-0270
Publisher
Elsevier
Publication Date
Volume
150
Issue
2
Identifiers
DOI: 10.1016/j.jneumeth.2005.06.010
Keywords
  • Auditory Brainstem
  • Inferior Colliculus
  • Phospho-Creb
  • Phospho-Erk
  • Phospho-Sap/Jnk
  • Western Blotting
Disciplines
  • Biology

Abstract

Abstract During attempts to quantify levels of phosphorylated cAMP response element binding protein (CREB-P) in guinea pig brain stem auditory nuclei by Western blotting, we compared the decay of CREB-P levels when tissues were homogenized in traditional Lysis buffer containing detergents or in 50 mM Tris–HCl buffer containing 0.32 M sucrose. The decay of CREB-P levels was retarded considerably in the Tris–Sucrose medium as compared to the Lysis buffer. Similarly, the levels of two other phospho-proteins, extracellular regulated kinases (ERK1/2-P) and stress activated protein/Jun-N-terminal kinase (SAP/JNK-P), were better preserved by the Tris–Sucrose medium. These findings imply that the detergents typically present in the Lysis buffer may disrupt organelles and increase the exposure of soluble phospho-proteins to hydrolyzing enzymes. In contrast, such exposure was probably minimized in the Tris–Sucrose medium, which is thought to preserve organelle integrity.

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