Panel of synaptic protein ELISAs for evaluating neurological phenotype.
The purpose of this study was to develop ELISAs for key neural proteins, three synaptic and one glial, that exist in different intracellular compartments, which would be used as a measure of synaptic phenotype. These assays would be valuable to neurologically phenotype transgenic mouse models of human disease and also human disease itself using minimal amounts of post-mortem tissue. We showed that supernatant from crude brain tissue homogenates extracted in RIPA buffer containing 0.1% SDS bind to synaptophysin, synaptosome-associated protein of 25 kDa (SNAP-25), post-synaptic density-95 (PSD-95), and glial fibrillary acidic protein (GFAP) antibody pairs with high affinity and selectivity. Overall, RIPA + 0.1% SDS were more efficient than RIPA + 2% SDS or a buffer containing only 1% Triton-X-100. Diluting the brain extracts resulted in dose-dependent binding to the antibody pairs for each neural protein, with EC50s that varied from 8.6 microg protein for PSD-95 to 0.23 microg for GFAP. The assays were used to measure synaptic marker protein levels at various times during mouse development and GFAP in a model of disease accompanied by neuroinflammation. Comparison of ELISAs with Western blots by measuring marker levels in brain extract from developing mice showed a greater relative difference in values derived from ELISA. These ELISAs should be valuable to phenotype the synapse in neurological disease and their rodent models.
Gottschall
Paul E
PE
Department of Pharmacology and Toxicology, College of Medicine, University of Arkansas for Medical Sciences, Slot #611, 4301 West Markham Street, Little Rock, AR, USA. pegottschall@uams.edu
Ajmo
Joanne M
JM
Eakin
Autumn K
AK
Howell
Matthew D
MD
Mehta
Hina
H
Bailey
Lauren A
LA
D000818:Animals; D001921:Brain / Q000254:growth & development / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005904:Glial Fibrillary Acidic Protein / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism; D010641:Phenotype; D051381:Rats; D017207:Rats, Sprague-Dawley; D050600:SNARE Proteins / Q000235:genetics / Q000378:metabolism; D012680:Sensitivity and Specificity; D013569:Synapses / Q000235:genetics / Q000378:metabolism*; D016708:Synaptophysin / Q000235:genetics / Q000378:metabolism; D050825:Synaptosomal-Associated Protein 25 / Q000235:genetics / Q000378:metabolism
D005904:Glial Fibrillary Acidic Protein; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D050600:SNARE Proteins; D016708:Synaptophysin; D050825:Synaptosomal-Associated Protein 25