Bio-Rad MicroRotofor™ Cell Lysis Kits - Manual (Page 8)

7. Add 1 µl of β-mercaptoethanol per 100 µl of yeast
suspension buffer.
8. Vortex until the cell suspension becomes homogenous.
9. Flick the vial of lyticase enzyme to mix. Add 10 µl of
lyticase enzyme per 100 µl of yeast cell pellet. Gently
mix. Lyticase will hydrolyze poly-(beta-1,3-glucose) for
lysis of the yeast cell wall.
10. Incubate the cell suspension at 37°C for 30–60 min.
11. Centrifuge the suspension at 10,000 x g for 5 min.
Remove and discard the supernatant carefully, leaving
the spheroplast pellet in the tube.
Note: If the majority of the sample is mucous-like and
difficult to pipet, the spheroplasts may have lysed. Reduce
the incubation time or start with a fresh culture.
12. Optional wash step: Add 600 µl of yeast suspension
buffer to the spheroplast pellet. Resuspend the
spheroplasts by gently tapping the tube. Centrifuge
again as above and discard the supernatant.
Note: This step washes away the lyticase from the protein
sample. Including this wash step may compromise the
yeast protein yield. Should you choose to eliminate this
step, you may anticipate the migration of lyticase on a 2-D
gel by knowing its pI (6.33) and molecular weight (54.6 kD).
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