Department of Biological Chemistry, Weizmann Institute of Science
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Rishal, I., Rozenbaum, M., Fainzilber, M. Axoplasm Isolation from Rat Sciatic Nerve. J. Vis. Exp. (43), e2087, doi:10.3791/2087 (2010).
Isolation of pure axonal cytoplasm (axoplasm) from peripheral nerve is crucial for biochemical studies of many biological processes. In this article, we demonstrate and describe a protocol for axoplasm isolation from adult rat sciatic nerve based on the following steps: (1) dissection of nerve fascicles and separation of connective tissue; (2) incubation of short segments of nerve fascicles in hypotonic medium to release myelin and lyse non-axonal structures; and (3) extraction of the remaining axon-enriched material. Proteomic and biochemical characterization of this preparation has confirmed a high degree of enrichment for axonal components.
This protocol allows axoplasm isolation with minimization of glial and vascular tissue contamination. The method yields approximately 70-100 μg total axoplasm protein per one 8-10 week old Wistar rat.
1. Dissect Sciatic Nerves
2. Incubation, Washing and Elution
3. Representative Results
Figure 1. Western blot comparing axoplasm isolated by the manual squeeze method with axoplasm samples isolated as shown in this protocol. Note the reduced levels of albumin and GFAP, representing blood protein and glial cell contaminations, respectively. In contrast, axonal tubulin b3 is enriched in this preparation, indicating a high level of axonal proteins.
Biochemical and proteomic analyses have confirmed that this procedure reduces serum and glial cell contamination 3 as compared to previously described methods for axoplasm isolation by mechanical squeeze 1. We have used this axoplasm isolation protocol to explore dynein-based retrograde signaling after sciatic nerve injury2, and expect it to have utility in the exploration of many other processes in adult peripheral nerve, including axon-glia interactions 4.
No conflicts of interest declared.
Mouse anti-GFAP clone G-A-5 was from Sigma (G6171). Rabbit anti-Albumin was from Cedarlane (CLAG5140). Mouse anti-Tubulin β3 and rabbit anti-gERK were from Sigma (T2200 and M5670 respectively).