Prepare extraction buffer + Protease Inhibitors and keep tube on ice, prepare 5µl buffer/brain
Dissect brains and collect them in microtube with extraction buffer on ice, try to transfer as little liquid as possible with the brains
Use a 200µl pipet tip which got squeezed on the tip to create a very thin opening
Homogenize the brains by pipetting up and down until the brains are smashed
If it takes a long time to homogenize, put the sample on ice in between
Spin the homogenate 5min at 13000 rpm at 4¡C
Collect the Supernatant SN and transfer to a new tube, add 2x Sample Buffer SB + DTT
Incubate 5min at 100¡C
Freeze samples at -20¡C
Extraction Buffer:
10mM HEPES/NaOH pH 7.4
150mM NaCl
2mM EDTA pH 8.0
1% NP40
store at 4¡C
Protease Inhibitors:
Calbiochem Protease Inhibitor Set V, EDTA-free
100x solution, store at -20
add Protease Inhibitors freshly before each extraction