Materials and Reagents

Pipette tips
10 µl pipette tips (SARSTEDT, catalog number: 70.1130 )
200 µl pipette tips (SARSTEDT, catalog number: 70.760.002 )
1,000 µl pipette tips (SARSTEDT, catalog number: 70.762 )
Reaction tubes
1.5 ml tubes (SARSTEDT, catalog number: 72.706 )
2 ml tubes (SARSTEDT, catalog number: 72.691 )
Mice (Protocol has been tested with male and female C57Bl/6 mice of 8-52 weeks)
Sucrose (≥ 99.5%) (Sigma-Aldrich, catalog number: S0389 )
HEPES (≥ 99.5%) (Sigma-Aldrich, catalog number: H3375 )
Protease inhibitor cocktail (cOmplete^TM Mini EDTA-free EasyPack) (Roche Diagnostics, catalog number: 04693159001 )
Phosphatase inhibitor cocktail 3 (Sigma-Aldrich, catalog number: P0044 )
Sodium chloride (NaCl) (≥ 99.5%) (Carl Roth, catalog number: 3957 )
Potassium chloride (KCl) (≥ 99%) (Carl Roth, catalog number: P017 )
Di-Sodium hydrogen phosphate (Na₂HPO₄) (≥ 98%) (Carl Roth, catalog number: P030 )
Potassium di-hydrogen phosphate (KH₂PO₄) (≥ 99%) (Carl Roth, catalog number: 3904 )
Lysis buffer (pH 7.5) (see Recipes)
Phosphate-buffered saline (PBS) (see Recipes)

Equipment

Pipettes (Research 0.5-10 µl, 10-100 µl, 100-1,000 µl) (Eppendorf)
Heraeus Biofuge Stratos (Rotor) (Heraeus Holding, catalog number: 3332 )
Ultrasound sonicator (Emerson Electric, BRANSON, model: 150 )
Stirring device (Ingenieurbüro CAT M. Zipperer, model: R 50D )
Glass homogenizer (2 ml) (Carl Roth, catalog number: TT57.1 )
Teflon pestle (Carl Roth, catalog number: TT63.1 )

Procedure

Notes:

The following protocol has been used to extract crude synaptosomal and free mitochondria fractions from mouse brain samples from mouse models of Alzheimer’s disease (see Breyhan et al. [2009] and Saul and Wirths [2017]). The user might need to modify the amount of solutions (e.g., lysis buffer) depending on the particulate downstream application (e.g., Western blotting or ELISA). For Western blots, we recommend to use 20 to 50 µg of total protein per well. This procedure will yield crude synaptosomal fractions with concentrations of approx. 7-10 µg/µl and microsomal and soluble enzyme fractions of approx. 4-6 µg/µl.
For mouse brain preparation, animals were anesthetized by carbon dioxide (CO₂) and sacrificed by cervical dislocation. Tissues were carefully dissected and either directly used or kept at -80 °C until further use. No differences were noted in yield or quality of the protein fractions if fresh or frozen tissue has been used.

Mechanically homogenize fresh or deep-frozen brain hemispheres in a weight:volume ratio of 1:10 using cold lysis buffer (1,000 µl buffer per 100 mg tissue, see Recipes) with 10 strokes in a glass-Teflon homogenizer at 800 rpm (Figure 2).
Note: Our lab uses one brain hemisphere with cerebellum and olfactory bulb removed and flash frozen on a plate of dry ice, however the presence of cerebellum or olfactory bulb does not compromise the procedure. For convenience, we do not remove the meninges upon harvesting the tissue.

Figure 2. Motor-driven homogenizer
Transfer the homogenate to 1.5 ml reaction tubes.
Spin the samples at 1,000 x g for 10 min at 4 °C to separate nuclei (Pellet P1).
Note: Centrifuge should be pre-chilled to 4 °C.
Remove the supernatant (S1) and transfer to a new 1.5 ml reaction tube. The pellet (P1) can be discarded.
Spin the supernatant at 12,000 x g for 10 min at 4 °C and transfer the resulting supernatant (S2) containing the light membrane fraction and soluble enzymes to a new 1.5 ml reaction tube.
Resuspend the pellet (P2) containing crude synaptosomes and mitochondria in 500 µl PBS (see Recipes).
Homogenates can be stored at -80 °C until further use.

Data analysis

The protein fractions obtained by using this protocol can be used in down-stream applications like Western-blot. Successful enrichment of crude synaptosomes and mitochondria can be e.g., demonstrated by Western blot analysis using marker proteins like post-synaptic density protein 95 (PSD95) or cytochrome c oxidase (CoxIV) (Saul and Wirths, 2017) (Figure 3).

Figure 3. Exemplary Western blot using antibodies against PSD95 and COXIV with β-actin as loading control

Recipes

Lysis buffer (pH 7.5)
0.32 M sucrose
5 mM HEPES
cOmplete^TM protease inhibitor cocktail (1 tablet per 10 ml)
Phosphatase inhibitor cocktail 3 (use at 1:100 dilution)
Note: Can be stored at 4 °C for 1 month or at -20 °C for up to 3 months.
PBS (1x working solution pH 7.4)
137 mM NaCl
2.7 mM KCl
8 mM Na₂HPO₄
2 mM KH₂PO₄

Acknowledgments

Financial support of the Alzheimer Forschung Initiative e.V. is gratefully acknowledged. This protocol was adapted from Gurd et al. (1974) with modifications.

References

Breyhan, H., Wirths, O., Duan, K., Marcello, A., Rettig, J. and Bayer, T. A. (2009). APP/PS1KI bigenic mice develop early synaptic deficits and hippocampal atrophy. Acta Neuropathol 117(6): 677-685.
Gurd, J. W., Jones, L. R., Mahler, H. R. and Moore, W. J. (1974). Isolation and partial characterization of rat brain synaptic plasma membranes. J Neurochem 22(2): 281-290.
Saul, A. and Wirths, O. (2017). Endogenous Apolipoprotein E (ApoE) fragmentation is linked to amyloid pathology in transgenic mouse models of Alzheimer’s disease. Mol Neurobiol 54(1): 319-327.