Axoplasm isolation from peripheral nerve.
Localized changes in the composition of axonal cytoplasm (axoplasm) are critical for many biological processes, including axon guidance, responses to injury, neurite outgrowth, and axon-glia interactions. Biochemical and molecular studies of these mechanisms have been heavily focused on in vitro systems because of the difficulty of obtaining subcellular extracts from mammalian tissues in vivo. As in vitro systems might not replicate the in vivo situation, reliable methods of axoplasm extraction from whole nerve would be helpful for mechanistic studies on axons. Here we develop and evaluate a new procedure for preparation of axoplasm from rat peripheral nerve, based on incubation of separated short segements of nerve fascicles in hypotonic medium to separate myelin and lyse nonaxonal structures, followed by extraction of the remaining axon-enriched material. We show that this new procedure reduces serum and glial cell contamination and facilitates proteomic analyses of axonal contents.
Rishal
Ida
I
Department of Biological Chemistry, Weizmann Institute of Science, 76100 Rehovot, Israel.
Michaelevski
Izhak
I
Rozenbaum
Meir
M
Shinder
Vera
V
Medzihradszky
Katalin F
KF
Burlingame
Alma L
AL
Fainzilber
Mike
M
D000818:Animals; D001369:Axons / Q000378:metabolism* / Q000648:ultrastructure; D035843:Biomedical Research / Q000379:methods*; D015153:Blotting, Western; D003593:Cytoplasm / Q000378:metabolism* / Q000648:ultrastructure; D007038:Hypotonic Solutions; D008297:Male; D013058:Mass Spectrometry; D051381:Rats; D017208:Rats, Wistar; D012584:Sciatic Nerve / Q000378:metabolism* / Q000648:ultrastructure; D013347:Subcellular Fractions / Q000378:metabolism* / Q000648:ultrastructure
D007038:Hypotonic Solutions