Yeast Protein extracts in TCA
This protocols allows for protein extraction in denaturing conditions, therby it is best used to preserve the post translational modifications of proteins
Materials:
- 20% TCA (Trichloroacetic Acid)
- 5% TCA
- 6X protein Blue
- Acid-washed glass beads (400-600 μm)
- 2M Tris-Base
- 1.5 and 2 ml Eppendorf tubes
Method:
- Start from ~10 ml of an exponentially growing culture at a concentration of ~8⋅106 cell/ml or equivalent
- Spin down the cells 2 minutes @ 4000 rpm in 50 ml tubes
- Resuspend the pellets in 1 ml of 20% TCA
- Transfer in 2 ml tubes
- Wash the pellets with another ml of 20% TCA
- Spin down at maximum speed for 1′ and discard the supernatant
- Resuspend in 50 μl of 20%TCA, vortex
- Add glass beads up to the meniscus using a measuring spoon
- Vortex in parallel 4’- 5′
- Add 100 μl 5% TCA and mix by vortexing
- Transfer the protein suspension to a new 1.5 ml tube using a p200
- Centrifuge 10′ at 3000 rpm
- Discard the supernatant (aspire with a vacuum pump)
- Add 100 μl of 2X PROTEIN BLUE (diluted from the 6X stock), the solution turns yellow
- Resuspend the pellet by thorough vortexing
- Add 50 μl of 2M Tris Base to neutralise the pH. Make sure the solution turns blue
- Boil 3’ a 95°C
- Centrifuge at max speed for 2’
- Transfer the superrnatant to a new 1.5 ml tube
- Store at -20°C for up to two weeks, -80°C for longer term storage