Total protein extraction

For Western from plant tissue

 

  1. Grind sample to a fine powder with liquid nitrogen
  2. Add two volume of extraction buffer, keep on ice 5 minutes
  3. Spin at 13krpm/10 minutes
  4. Transfer sup a clean tube
  5. Repeat spin once and transfer sup to a clean tube
  6. Mix with protein loading buffer and boil 5 minutes.

 

Extraction buffer:

100 mM Tris-HCl pH8

0.1% SDS

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.