Manual

and/or further lyse some of the spheroplasts. Should you
choose to eliminate this step, you may anticipate the
migration of lysozyme on a 2-D gel by knowing its pI (9.32)
and molecular weight (14.3 kD).
12. Add 500 µl of freshly prepared PSB solution to the
spheroplast pellet.
13. Sonicate the suspension with an ultrasonic probe to
break down the cell membrane and genomic DNA.
Sonication should be performed in an ice bath to
prevent heating. Sonication should be performed with
bursts of 10–15 sec, with chilling of the suspension on
ice between ultrasonic bursts.
14. Centrifuge at 20,000 x g for 30 min at 20°C and collect
the clear lysate.
15. Resuspend the residual cell debris in 250 µl of PSB
solution. Sonicate the suspension once briefly. Repeat
the centrifugation in step 14, collect the supernatant,
and pool with the first supernatant.
16. Determine the protein concentration of the extract.
This is best done using the RC DC protein assay
(catalog #500-0121 or 500-0122), which is compatible
with the detergents and reducing agents present in
PSB. If performing the RC DC protein assay, keep in
mind that two washes of the sample are recommended.
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