Manual

Note: For best results, use a wet cell pellet from a freshly

grown bacterial culture. Do not use frozen or stored stocks.

7. Gently flick the bottom of the tube to resuspend the

cell pellet (no longer than 1 min). If the cell pellet doesn't

resuspend readily, the cells may no longer be viable for

spheroplast preparation. Use fresh cultures.

8. Mix the lysozyme by gently flicking the vial and add it

to the sample (5 µl of lysozyme per 50 µl of wet cell

pellet). Mix gently.

9. Incubate the suspension at 37°C for 60 min.

10. Centrifuge the suspension at 5,000 x g for 10 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.

11. Optional wash step: Add 500 µl of bacteria 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 lysozyme from the

protein sample. Including this wash step may slightly

compromise the bacterial protein yield since it may remove

any bacterial proteins existing outside the spheroplast wall

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