Immunoblotting patterns of cytoskeletal dendritic protein expression in human neocortex.
Qualitative and quantitative evaluations of cytoskeletal proteins are critical for understanding physiological and pathological processes affecting the nervous system. Most of such studies on human samples have only used immunohistochemical techniques. We describe a complementary immunoblotting approach, for the assessment of neuronal cytoskeletal proteins, which employs fresh frozen postmortem tissues. We found that cytosolic fractions are suitable for qualitative and quantitative evaluations of four major dendritic cytoskeletal proteins: microtubule-associated protein (MAP)-2, MAP-5, and high- and medium-molecular-weight nonphosphorylated neurofilaments. The enhanced chemiluminescence (ECL) technique revealed consistent and distinctive immunoblotting patterns for all four proteins in both monkey (no postmortem delay) and human (17-34 h postmortem interval) samples, some of which differed from those found in rodents. Quantitations of blots, by tissue protein-optical density curves that demonstrated linearity of the measurements in the 0- to 100-microgram range, support the feasibility of these immunoassays for the study of neurologic disorders.
Kaufmann
W E
WE
Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD, USA. wekaufma@welchlink.welch.jhu.edu
Taylor
C V
CV
Lishaa
N A
NA
D000818:Animals; D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D003599:Cytoskeleton / Q000378:metabolism*; D003712:Dendrites / Q000378:metabolism*; D006801:Humans; D008163:Luminescent Measurements; D008251:Macaca; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D008970:Molecular Weight; D019579:Neocortex / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D016900:Neurofilament Proteins / Q000378:metabolism
D000911:Antibodies, Monoclonal; D008869:Microtubule-Associated Proteins; D009419:Nerve Tissue Proteins; D016900:Neurofilament Proteins; C085284:microtubule-associated protein 1B