Tris-base buffer
Tris-base & detergent buffer
Na-Acetate buffer
Additional compounds to protect proteins and state of phosporylation
General extraction procedure
 

Tris-base buffer

50mM TRIS-base
5mM MgCl₂
250mM Sucrose
2mM ATP (fresh)
1mM DTT (fresh)

pH to 7.4

Tris-base & detergent buffer

50mM TRIS-base
150mM NaCl
1% Triton-X100

pH to 7.4

Comment: used for western blot with phospospecific antibodies. Combined with NaF, Na₃VO₄ and protease inhibitors

Na-Acetate buffer

50mM Na-Acetate
0.1% Triton-X100

pH to 7.4

Comment: used for routine protein extraction in e.g. the Shc-paper (Jiang 2003).

Additional compounds to protect proteins and state of phosporylation

1mM NaF (MW 41.99)
1mM Na₃VO₄ (sodium-ortho-vanadate) (MW 183.9)
Complete inhibitor tablets (protease inhibitor cocktail witout EDTA (Roche cat #1 873 580)) 1 tablet/50ml

Prepare small amount of 500X stock solutions:
1ml stocks
500mM NaF      0,020995g
500 mM Na₃VO₄  0,09195g

Add 100µl of each stock solution to 50ml lysis buffer just prior to use.
Add 1 inhibitor tablet to 50ml of lysis buffer just prior to use
 

General extraction procedure

Keep all samples on ice throughout the extraction to prevent protein degradation.
Homogenize tissue.
Add 100µl of lysis buffer per 30-50mg tissue.
Allow samples to incubate 1h on ice when using detergent lysis.
Centrifuge 13 000rpm (?) for 15min at 4ºC
Collect supernatant
Store at -20ºC