Assaying multiple biochemical variables from the same tissue sample.
Experiments often involve multiple analyses, such as assays of neurotransmitters and proteins, and this can require different initial sample preparations. Typically, this is accomplished by using different animals or different tissue samples from the same animal. Either approach renders comparisons between assays more variable and greatly increases the effort and/or cost. Using tissue collected from rat striatum and molecules of special relevance to studies of Parkinson's disease, we show that tissue sonication in water prior to aliquoting into the appropriate concentrated solutions (e.g. HClO(4) and lysis buffers) permits several types of measurements to be made from the same initial samples. Dopamine and its metabolite homovanillic acid, serotonin and its metabolite 5-hydroxyindoleacetic acid, tyrosine hydroxylase and its phosphorylation at Ser19 and Ser31, and the dopamine transporter were unaffected. However, phospho-Akt levels fell slightly and phospho-ERK1/2 tended to drop. We also present a simple technique to preserve phosphorylation state of proteins such as ERK1/2 by perfusing animals through the heart with a phosphatase inhibitor, NaF. Dopamine metabolite dihydroxyphenyl acetic acid (DOPAC) levels were raised with both techniques, however. The general principles reported here are likely to apply to other brain regions, facilitate multiple comparisons of variables, increase efficiency, and decrease costs.
Leak
Rehana K
RK
Pittsburgh Institute for Neurodegenerative Diseases, Department of Neurology, 3501 Fifth Ave, 7026 BST3, University of Pittsburgh, Pittsburgh, PA, United States. leakr@duq.edu
Castro
Sandra L
SL
Jaumotte
Juliann D
JD
Smith
Amanda D
AD
Zigmond
Michael J
MJ
D000818:Animals; D001681:Biological Assay / Q000379:methods; D001921:Brain / Q000378:metabolism*; D001923:Brain Chemistry / Q000502:physiology*; D003342:Corpus Striatum / Q000737:chemistry / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D008297:Male; D009448:Neurochemistry / Q000379:methods*; D010766:Phosphorylation / Q000502:physiology; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D013010:Sonication / Q000379:methods*
D004791:Enzyme Inhibitors; D051057:Proto-Oncogene Proteins c-akt; D048049:Extracellular Signal-Regulated MAP Kinases