Brain extracts from Xenopus tadpoles

 

 

Prepare extraction buffer + Protease Inhibitors and keep tube on ice, prepare 5l 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 200l 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 4C

 

Collect the Supernatant SN and transfer to a new tube, add 2x Sample Buffer SB + DTT

 

Incubate 5min at 100C

 

Freeze samples at -20C

 

 

Extraction Buffer:

 

10mM HEPES/NaOH pH 7.4

150mM NaCl

2mM EDTA pH 8.0

1% NP40

 

store at 4C

 

 

Protease Inhibitors:

Calbiochem Protease Inhibitor Set V, EDTA-free

100x solution, store at -20

add Protease Inhibitors freshly before each extraction