Phospho-CREB and other phospho-proteins: improved recovery from brain tissue.
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.
Suneja
Sanoj K
SK
Department of Neuroscience, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT 06030-3401, USA. ssuneja@neuron.uchc.edu
Mo
Zhicheng
Z
Potashner
Steven J
SJ
D000818:Animals; D015153:Blotting, Western; D001923:Brain Chemistry; D001933:Brain Stem / Q000378:metabolism*; D019411:Clinical Laboratory Techniques; D017362:Cyclic AMP Response Element-Binding Protein / Q000032:analysis* / Q000302:isolation & purification*; D006168:Guinea Pigs; D010766:Phosphorylation; D011506:Proteins / Q000032:analysis / Q000302:isolation & purification; D014325:Tromethamine
D017362:Cyclic AMP Response Element-Binding Protein; D011506:Proteins; D014325:Tromethamine