Journal
JOURNAL OF NEUROSCIENCE METHODS
Volume 150, Issue 2, Pages 238-241Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.jneumeth.2005.06.010
Keywords
auditory brainstem; inferior colliculus; phospho-CREB; phospho-ERK; phospho-SAP/JNK; western blotting
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Funding
- NIDCD NIH HHS [DC00199] Funding Source: Medline
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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. (c) 2005 Elsevier B.V. All rights reserved.
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