Blot and culture analysis of neuronotrophic factors in nerve regeneration chamber fluids.
The fluid accumulating in silicone nerve regeneration chambers implanted between the cut ends of rat sciatic nerve contains neuronotrophic activities towards embryonic chick ciliary and sympathetic neurons. The blot and culture technique of Carnow et al. was used to determine if part of the neuronotrophic activities is due to ciliary neuronotrophic factor, which supports the survival of both types of neurons in vitro. The technique involves separating the fluid proteins by SDS-polyacrylamide gel electrophoresis, Western transfer, and then culturing of purified neurons on the nitrocellulose blots. After 24 hr surviving neurons are restricted to regions of the blot where neuronotrophic factor is present. Analysis of 1 and 2 day fluids showed that a multitude of factors are present, particularly in the 19-30 kD molecular weight range, with discrete peaks of activity at molecular weights consistent with those reported for ciliary neuronotrophic factor. There were several other peaks of activity present in the fluids in addition to these.
Bates
D J
DJ
University of Adelaide Department of Paediatrics, Adelaide Medical Centre for Women and Children, South Australia.
Ranford
J A
JA
Mangelsdorf
D C
DC
D000818:Animals; D015153:Blotting, Western; D002470:Cell Survival; D002478:Cells, Cultured; D020934:Ciliary Neurotrophic Factor; D003101:Collodion / Q000737:chemistry; D003470:Culture Media; D015194:Diffusion Chambers, Culture; D004591:Electrophoresis, Polyacrylamide Gel; D005123:Eye / Q000294:innervation; D005260:Female; D005728:Ganglia, Sympathetic / Q000166:cytology; D009414:Nerve Growth Factors / Q000032:analysis*; D009416:Nerve Regeneration / Q000502:physiology*; D009419:Nerve Tissue Proteins / Q000032:analysis*; D009474:Neurons / Q000166:cytology*; D051381:Rats; D011919:Rats, Inbred Strains; D012584:Sciatic Nerve / Q000293:injuries
D020934:Ciliary Neurotrophic Factor; D003470:Culture Media; D009414:Nerve Growth Factors; D009419:Nerve Tissue Proteins; D003101:Collodion