Quantitative analysis of GAP-43 expression by neurons in microcultures using cell-ELISA.
A cell-ELISA technique is described which allows the quantification of GAP-43 protein in a large number of microcultures of adult dorsal root ganglion neurons. GAP-43 is measured in the 1-10 ng range, corresponding to the amount of GAP-43 present in fewer than 500 DRG neurons. Specificity of the assay is confirmed using Western blotting and immunocytochemistry. The GAP-43 content of adult DRG microcultures rises during 2 weeks in culture, although the number of surviving neurons decreases. The GAP-43 content of cultured adult DRG neurons is not increased by chronic exposure to added nerve growth factor after 7 days in vitro. However, GAP-43 is increased in DRG taken from animals with prior peripheral nerve injury, and is decreased by chronic exposure to dibutyryl cyclic AMP after 7 days in vitro. The method affords the sensitivity and statistical power to document modest changes in GAP-43 protein abundance in complex cultures.
Schreyer
D J
DJ
Department of Physiology, Queen's University, Kingston, Canada.
Andersen
P L
PL
Williams
K
K
Kosatka
I
I
Truong
T N
TN
D000818:Animals; D000918:Antibody Specificity; D015153:Blotting, Western; D002452:Cell Count; D002470:Cell Survival / Q000502:physiology; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D019922:GAP-43 Protein; D005727:Ganglia, Spinal / Q000166:cytology; D007124:Immunoenzyme Techniques; D008562:Membrane Glycoproteins / Q000032:analysis* / Q000096:biosynthesis / Q000276:immunology; D008869:Microtubule-Associated Proteins / Q000032:analysis; D009419:Nerve Tissue Proteins / Q000032:analysis* / Q000096:biosynthesis / Q000276:immunology; D009474:Neurons / Q000737:chemistry* / Q000166:cytology / Q000378:metabolism; D051381:Rats; D013997:Time Factors
D019922:GAP-43 Protein; D008562:Membrane Glycoproteins; D008869:Microtubule-Associated Proteins; D009419:Nerve Tissue Proteins; D000242:Cyclic AMP