Brain Synaptosomal Preparation
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实验试剂
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)
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.
