Sample analysis

1. Preparation of soluble and insoluble cell extracts from B. subtilis
- harvest cells by centrifugation (10 min, 6,000 x g, 4 °C)
- wash and resuspend in 50 mM sodium phosphate buffer (pH 7.0) at an OD600 of 10
- disrupt cells by ultrasonication (12 W, 6 x 15 pulses with 15 sec intervals) in 1.5 ml
Eppendorf tubes containing 1 ml of cell suspension, supplemented with lysozyme
(250 μg/μl, CB-0663-5GAM), on ice.
- alternatively, cells can be disrupted by beat beating:
disrupt three times with glass beads (0.1 mm in diameter) (1 g/ml of cell suspension) in
an orbital mixer at 180 V, with the mix kept on ice for 3 min between each disruption
- take 100 μl of the preparation as first total protein sample (T1)
- remove cell debris by centrifugation at 4,300 x g, 10 min
- take 100 μl of the supernatant for the second total protein sample (T2)
- spin at 8.200 x g (10 min, 4 °C)
to separate into insoluble (I) and soluble (S) protein fractions.
- per sample use the amount of protein corresponding to 0.025 of OD600 for separation by SDS-PAGE
- analyze samples by immunoblotting with specific antiserum