实验概要

The protocol provides a method of brain synaptosomal preparation.

主要试剂

1. 2.55 M sucrose (unbuffered)

    1) Heat on a stirring block to dissolve, then bring up to 1 L.

    2) Determine concentration using a refractometer and adjust to  2.55 M sucrose (refractive index is 1.4558 for 87.28% sucrose (w/v) at  2.5498 M)

2. 10% NP-40 STEN lysis buffer (-BSA)

    1) 5 ml 2x STEN

    2) 1 ml NP-40

    3) 0.1 ml 100X PI

    4) 0.1 ml 100x Pefabloc

    5) 3.8 ml ddH₂O

    6) Store at -20°C.

实验步骤

1. Homogenize  brain tissue. Add 5 ml of 0.32 M sucrose. Homogenize with 12 strokes of a  19x84mm tissue grinder (Potter Elvehjem, plastic coated) at 800 rpm.

2. Centrifuge to remove large debris. Centrifuge for 10 min at 1000 g at RT. Remove 100 ul for synaptophysin analysis.

3. Layer supernatant onto sucrose and centrifuge again.

4. Carefully layer the supernatant onto 4 ml of 1.2 M sucrose in a SW41 centrifuge tube (Beckman).

5. Spin at 160,000 g for 15 min (= 33,000 rpm with SW41 rotor).

6. Carefully remove synaptosome layer. The synaptosomes are at the  interface between the 1.2 M and 0.32 M sucrose layers. It is a slightly  cloudy thin layer. Mitochondria and lysosomes pellet to the bottom.

7. Remove 100 ul for synaptophysin analysis.

8. Dilute synaptosomes with 0.32 M sucrose. Add 4 ml of 0.32 M  sucrose to synaptosomes, mix, and then carefully layer onto 4 ml of 0.8 M  sucrose in a fresh centrifuge tube.

9. Centrifuge to pellet synaptosomes.

10. Spin at 160,000 g for 15 min. The pellet is enriched in synaptosomes.

11. Discard the supernatant and resuspend in 1 ml of 1X STE.

12. Remove 50 ul for electron microscopy analysis.

13. Lyse synaptosomes for immunoprecipitation analysis. Add 110 ul of  10% NP-40 STEN lysis (- BSA) and nutate at 4°C for 20 min. Protein  normalize samples, remove sample for synaptophysin analysis then  continue IP according to standard protocols.