Total protein extraction
For Western from plant tissue
- Grind sample to a fine powder with liquid nitrogen
- Add two volume of extraction buffer, keep on ice 5 minutes
- Spin at 13krpm/10 minutes
- Transfer sup a clean tube
- Repeat spin once and transfer sup to a clean tube
- Mix with protein loading buffer and boil 5 minutes.
100 mM Tris-HCl pH8
Add fresh: 2% beta-mercaptoethanol and protease cocktail inhibitor 1X (cocktail is optional).
When protein extracts are stored in the freezer before being loaded they should be warmed up at 40C for 2-3 minutes to re-solubilize the SDS.
**Protein amount can be quantified with Bradford.