Synaptic Plasma Membrane Prep

All procedures should be done at 4 degrees Celsius using precooled reagents. For rat and mouse samples, immediately remove the brain from the cranium into ice cold HEPES-buffered sucrose (0.32 M sucrose, 4 mM HEPES pH 7.4) containing a freshly added protease inhibitor cocktail (required, see below) and phosphatase inhibitor cocktail (optional, see below). Different parts of the brain can be subdissected and enriched plasma membranes/PSD can be prepared as described below.

Protease inhibitors
PMSF (0.1 mM)
Aprotinin (1.5 µg/ml ea.)
Antipain / leupeptin (10 µg/ml ea.)
Chymostatin / pepstatin (10 µg/ml ea)
Benzamidine (0.1 mg / ml)

Phosphatase inhibitors
EGTA (2 mM)
NaF (50 mM )
sodium pyrophosphate (10 mM)
β-glycerophosphate (20 mM)
para-nitrophenylphosphate (PNPP) (1 mM)
microcystin LR (optional) (1 mM)
Ammonium molybdate (0.1 mM)

1. Add 10 volumes of HEPES-buffered sucrose (0.32 M sucrose, 4 mM HEPES pH 7.4) to the tissue and homogenize in a motor driven glass-teflon homogenizer at ~900 rpm (10-15 strokes). Never use a polytron.

2. Centrifuge the homogenate (Hom.) at 800-1000 x g at 4 degrees Celsius to remove the pelleted nuclear fraction (P1).

3. Spin resulting supernatant (S1) at 10,000 x g for 15 min (9200 rpm in SL50T rotor) to yield the crude synaptosomal pellet (P2)

4. Resuspend pellet (P2) in 10 volumes of HEPES-buffered sucrose and then respin at 10,000 x g for another 15 min to yield the washed crude synaptosomal fraction (P2’).

5. Lyse resulting pellet by hypoosmotic shock in 9 volumes ice cold H20 plus protease / phosphatase inhibitors and three strokes of glass-teflon homogenizer

6. Rapidly adjust to 4 mM HEPES using 1 M HEPES, pH 7.4 stock solution.

7. Mix constantly in cold room for 30 min to ensure complete lysis

8. Centrifuge lysate at 25,000 x g for 20 min (14,500 rpm in SL50T rotor) to yield a supernatant (S3, crude synaptic vesicle fraction) and a pellet (P3, lysed synaptosomal membrane fraction).

9. Resuspend P3 pellet in HEPES-buffered sucrose.

10. Using a Pasteur pipette, layer the resuspended membranes on top of a discontinuous gradient containing 0.8 to 1.0 to 1.2 M sucrose (top to bottom; equals 27% / 34% / 41%) in a clear tube. Don’t forget to add protease / phosphatase inhibitors to the sucrose solutions! Always use a Pasteur pipette to pour layer and add sample.

11. Centrifuge the gradient at ~150,000 x g for 2 hr in a swinging bucket rotor (30,000 rpm in SW41 Ti; 36,000 rpm in SW50.1 Ti; 42,000 rpm in TLS-55; 28,000 rpm in SW28).

12. Recover synaptic plasma membranes in the layer between 1.0 and 1.2 M sucrose

13. Dilute to 0.32 sucrose by adding 2.5 volumes of 4 mM HEPES pH 7.4.

14. Pellet by centrifugation at 150,000 x g for 30 min. 42,000 rpm in 70.1 Ti; 55,000 rpm in TLA 100.3.

15. Resuspend resulting pellet (SPM) in PBS (pH 7.4) or (50 mM HEPES pH 7.4, 2 mM EDTA) with protease and phosphatase inhibitors.

16. Can store at -80 degrees Celsius.

Postsynaptic Densities

For references, see Carlin et al. (1980) and Cho et al. (1992).

1. Resuspend synaptic plasma membranes prepared as above in 3-5 ml of ice cold 50 mM HEPES pH 7.4, 2 mM EDTA, plus protease / phosphatase inhibitors. Add Triton X-100 to 0.5%

2. Rotate in cold room for 15 min.

3. Centrifuge at 32,000 x g for 20 min (22,000 rpm in 70.1 Ti; 28,000 rpm in TLA 100.3) to obtain the PSD-1T pellet.

4. Resuspend PSD-1T in 3 mL ice-cold 50 mM HEPES pH 7.4, 2 mM EDTA plus protease / phosphatase inhibitors). Save 1 mL.

5. To half of the remaining resuspended pellet (~1 mL), add Triton X-100 to 0.5% and rotate in cold room again for 15 min.

6. Centrifuge at 200,000 x g for 20 min to obtain the PSD-2T pellet. 50,000 rpm in 70.1 Ti; 65,000 rpm in TLA 100.3.

7. In a separate experiment resuspend the second half of the PSD-1T pellet and incubate for 10 min in ice cold 3% sarcosyl (same as N-lauroyl sarcosine) in 50 mM HEPES pH 7.4, 2 mM EDTA, plus protease / phosphatase inhibitors.

8. Centrifuge at 200,000 x g for 1 hour to obtain the PSD-1T+S pellet.

9. All pellets can be resuspended in PBS or 50 mM HEPES pH 7.4, 2 mM EDTA plus protease / phosphatase inhibitors or SDS-PAGE sample buffer.

10. For PSD-2T pellets, may need some SDS (0.2%) to dissolve completely.



Synaptic Vesicle Prep

1. To prepare synaptic vesicles, prepare S3 fraction as in Synaptic Plasma Membrane Prep above.

2. Centrifuge S3 fraction at 165,000 x g for 2 hours at 55,000 rpm in TLA 100.3, 45,000 rpm in 70.1 Ti

3. Resuspend in PBS or 50 mM HEPES pH 7.4, 2 mM EDTA plus protease / phosphatase inhibitors.

Pure Synaptosome Prep

For crude synaptosomes, can simply use P2’ fraction from the Synaptic Plasma Membrane Prep.

For added purity:

1. Layer P2’ fraction onto 4 ml of 1.2 M sucrose

2. Centrifuge at 230,000 x g for 15 min in swinging bucket rotor. 38,000 rpm in SW 41 Ti, 44,000 rpm in SW50.1, 52,000 rpm in TLS-55

3. Collect gradient interphase.

4. Dilute to ~7-8 ml with ice-cold HEPES-buffered sucrose (0.32 M sucrose, 4 mM HEPES pH 7.4)

5. Layer onto 4 ml of 0.8 M sucrose

6. Centrifuge at 230,000 x g for 15 min

7. Pellet contains pure synaptosomes.

8. For bioassays, wash and resuspend in appropriate incubation buffer:

For electroporation or functional release assays: (140 mM NaCl, 5 mM KCl, 5 mM NaHC03, 1.2 mM Na2 HPO4, 1 mM MgCl2, 20 mM HEPES ph 7.4, 10 mM dextrose (added fresh), +- 1.8 mM CaCl2, and optional ATP regeneration system.

For digitonin permeabilization: 115 mM KOAc, 25 mM HEPES, pH 7.4, 5 mM NaOAc, 0.05 mM EGTA containing 0.025% digitonin, an ATP regeneration system, and 10 mM DTT. (see Meacham, Patterson, et al., Nature Cell Biology, 2001)

ATP regeneration system – 5mM Mg-ATP, 80 mM creatine phosphate, 0.5 mg/ml creatine phosphokinase.

9. Otherwise, can solubilize as desired, or subfractionate further into synaptosomal membranes and synaptic vescles by hypotonic lysis as described under Synaptic Plasma Membrane Prep