- 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.
- Centrifuge to remove large debris. Centrifuge for 10 min at 1000 g at RT. Remove 100 ul for synaptophysin analysis.
- Layer supernatant onto sucrose and centrifuge again.
- Carefully layer the supernatant onto 4 ml of 1.2 M sucrose in a SW41 centrifuge tube (Beckman).
- Spin at 160,000 g for 15 min (= 33,000 rpm with SW41 rotor).
- 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.
- Remove 100 ul for synaptophysin analysis.
- 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.
- Centrifuge to pellet synaptosomes.
- Spin at 160,000 g for 15 min. The pellet is enriched in synaptosomes.
- Discard the supernatant and resuspend in 1 ml of 1X STE.
- Remove 50 ul for electron microscopy analysis.
- 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.
2.55 M sucrose (unbuffered)
- Heat on a stirring block to dissolve, then bring up to 1 L.
- 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)
10% NP-40 STEN lysis buffer (-BSA)
- 5 ml 2x STEN
- 1 ml NP-40
- 0.1 ml 100X PI
- 0.1 ml 100x Pefabloc
- 3.8 ml ddH2O
- Store at -20°C.
amyloid beta in brain -
if value = 20 pM
then 20 pmole/Liter = 20 pmole/1000g
so x 12 for dilution = 240 pmole/1000g = 0.24 pmol/g