A method to prepare translationally active, intact synaptoneurosomes (SNs) from mouse brain cortex is described. The method uses a discontinuous Percoll-sucrose density gradient allowing for the quick preparation of active SNs.
Synaptoneurosomes (SNs) are obtained after homogenization and fractionation of mouse brain cortex. They are resealed vesicles or isolated terminals that break away from axon terminals when the cortical tissue is homogenized. The SNs retain pre- and postsynaptic characteristics, which makes them useful in the study of synaptic transmission. They retain the molecular machinery used in neuronal signaling and are capable of uptake, storage, and release of neurotransmitters.
The production and isolation of active SNs can be problematic using medias like Ficoll, which can be cytotoxic and require extended centrifugation due to high density, and filtration and centrifugation methods, which can result in low activity due to mechanical damage of the SNs. However, the use of discontinuous Percoll-sucrose density gradients to isolate SNs provides a rapid method to produce good yields of translationally active SNs. The Percoll-sucrose gradient method is quick and gentle as it employs isotonic conditions, has fewer and shorter centrifugation spins and avoids centrifugation steps that pellet SNs and cause mechanical damage.
The discontinuous Percoll-sucrose gradient preparation described herein is a quick, reliable method to isolate active SNs, which can be used in a variety of synaptic transmission experiments. This gradient method, which is based on the method developed by Dunkley et al.,3,4 is a subcellular brain fractionation procedure that isolates both pre- and postsynaptic membrane-derived vesicles that are associated with one another. Without further purification these SNs are compatible with a variety of molecular biolog…
The authors have nothing to disclose.
We would like to thank B. K. August from the University of Wisconsin-Madison Electron Microscope Facility for the electron microscopy. This work was supported by NIH grants R01-DA026067 and P30-HD03352 (to J.S.M.).
Name of the reagent | Company | Catalogue number | Comments (optional) |
---|---|---|---|
Micro BCA Protein Assay Kit | Pierce | 23235 | |
CaCl2 | Fisher | C79-500 | |
CO2 gas | Airgas (UW-MDS) | CD 50 | |
EDTA | RPI | E57020 | |
EtOH | Fisher | A407SK-4 | |
HCl | Fisher | A142-212 | |
Percoll | GE Healthcare | 17-0891-01 | |
KH2PO4 | Fisher | P285-500 | |
PierceSDS-PAGE Sample Prep Kit | Pierce | 89888 | |
NaCl | RPI | S23020 | |
NaHCO3 | Fisher | BP328-500 | |
Na2PHO4 | Fisher | S381-500 | |
Sucrose | RPI | S24060 | |
Tris Base | RPI | T60040 | |
Tetrodotoxin | Sigma | T5651 | |
Express Pro Label Mix S35 Easy Tag | Perkin Elmer | NEG772 | |
Equipment | Company | Catalogue number | Comments (optional) |
Dissection tools | |||
Dounce homogenizer, 7 mL (comes with two glass pestles labled ” A” and “B”) | Wheaton | ||
P1000 Gilson Pipetman | Gilson | F123602 | |
Allegra 6KR Centrifuge | Beckman Coulter | 366830 | |
GH 3.8 Rotor, Swinging bucket rotor | Beckman Coulter | 360581 | |
Beckman J2-21 Centrifuge | Beckman | ||
Beckman tubes with caps | Beckman | 355672 | |
White walled adapters | Beckman | 342327 | |
Blue walled adapters | Beckman | ||
JA-17 Rotor, Fixed-angle rotor | Beckman | 369691 |
Table of antibodies used for western blots:
Name of Antibody | Company | Catalogue number | Host Species | Dilution Factor |
---|---|---|---|---|
β-Actin | Sigma | A5441 | mouse | 1:2000 |
GFAP | Santa Cruz | sc-65343 | mouse | 1:200 |
GP73 | Santa Cruz | Sc-134509 | rabbit | 1:200 |
HSC70 | Santa Cruz | sc-7298 | mouse | 1:200 |
Laminβ | Santa Cruz | Sc-6261 | goat | 1:200 |
Prohibitin | Santa Cruz | sc-28259 | rabbit | 1:200 |
PSD95 | Millipore | MAB1596 | mouse | 5 μg/μL |
SNAP25 | AbCam | ab5666-100 | rabbit | 1:2000 |
Synaptophysin | Millipore | MAB368 | mouse | 1:500 |
β-3-Tubulin | Santa Cruz | sc-80016 | mouse | 1:200 |