Alkaline Lysis Protocol for Isolating Protein from S. cerevisiae

Alkaline Lysis Protocol for Isolating Protein from S. cerevisiae

1. To the cell pellet add 200ul of 0.1N NaOH. Vortex to resuspend the pellet.

2. Incubate at room temperature for 20-30mins

3. Spin down at 12,000rpm for 5 mins and discard supernatant (by pipetting).

4. To the pellet, add desired amount of lysis buffer (about 70-200ul depending upon the size of the pellet)

5. vortex to resuspend the pellet. boil at 95⁰C for 7-8mins.

6. Spin down at 13,000 rpm for 15mins.

7. collect supernatant in a fresh microfuge tube. Estimate protein using BCA and load on gel.

Composition of lysis buffer (final conc) :

SDS - 2%

glycerol - 10%

1M Tris (ph 6.8) - 40mM