Materials and Reagents

B. subtilis
Trichloroacetic acid (Merck KGaA, catalog number: 1.00.807.0250 )
Acetone (J.T.Baker^®, catalog number: 9006-01 )
BCA-assay (Pierce Antibodies, catalog number: 23225 )
Casein Hydrolysate (Oxoid Limited, catalog number: LP0041 )
(CH)-medium (see Recipes)
Solution G (see Recipes)
Resuspension Buffer (see Recipes)

Equipment

50 ml flasks with baffles
Acrodisc^® syringe filters with a pore size of 0.2 µm (Pall, catalog number: 4652 )
Refrigerated centrifuge (e.g. Eppendorf, model: 5810R )

Procedure

Inoculate B. subtilis to an OD₆₀₀of 0.1 in appropriate medium [e.g. CH, lysogeny broth (LB), Spizizen minimal medium (SMM); here we used 10 ml cultures in CH-medium].
Grow cells to respective OD₆₀₀ (here we used an OD₆₀₀ of 4; cells growing at 37 °C; 150 rpm in 50 ml flasks with baffles).
Harvest cells by centrifugation (≥ 2,800 x g; 4 °C; 20 min).
Note: Keep cell pellet as a control for later SDS-PAGE analysis.
Transfer the supernatant into fresh tubes and centrifuge again (2,800 x g; 4 °C; 20 min).
Repeat step 4.
Collect the supernatant and sterile filter it using Acrodisc^® syringe filters with a pore size of 0.2 µm.
Add ice cold trichloroacetic acid [90% (w/v) stock] to the solution to a final concentration of 10% (v/v). This will result in precipitation of the proteins in the solution.
Harvest the precipitate by centrifugation (11,000 x g; 4 °C, 5 min). A small white pellet should be visible.
Wash the pellet twice with 2 ml of ice cold acetone (PA grade or higher quality) and centrifugation (11,000 x g; 4 °C, 5 min).
1. For quantitative analysis dry the pellet for 15 min at 60 °C to remove remaining traces of acetone. Furthermore, the pellet should be resuspended in H₂O instead of buffer (step 10).
Resuspend the pellet in resuspension buffer (we used a volume of 50 µl).
Note: The volume can be scaled up or down with respect to your amount of cells (secreted proteins).
The amount of secreted proteins can be analyzed by BCA-assay or analyzed by SDS-PAGE.
Note: As a control if the isolation has worked successfully the isolated secreted proteins and the pelleted cells (see step 3) should be resuspended in H₂O or buffer (5-10x volume of pelleted cells) and analyzed by SDS-PAGE. The band pattern of the isolated proteins and the cells should be different. If the band pattern is similar, your isolated proteins are likely contaminated by cells.

Recipes

CH-medium
50 ml solution G (see below)
0.05 ml 0.1 M CaCl₂
0.02 ml 1 M MgSO₄^.7H₂O
0.1 ml 0.0475 M MnSO₄
0.5 ml 2 mg/ml tryptophan
Sterilize by filtering using Acrodisc^® syringe filters; prepare freshly
Solution G
25.0 g casein hydrolysate
9.2 g L-glutamic acid
3.125 g L-alanine
3.48 g L-asparagine
3.4 g KH₂PO₄
1.34 g NH₄Cl
0.27 g Na₂SO₄
0.24 g NH₄NO₃
2.45 mg FeCl₃^.6H₂O
ddH₂O 2.35 L and adjust pH to 7.0 by using 10 N NaOH
Sterilize by autoclaving; stored at 4 °C
Resuspension Buffer
5 mM MgCl₂
50 mM Tris-HCl (pH 7.4)

Acknowledgments

This protocol is adapted from Bach and Bramkamp (2013).

References

Bach, J. N. and Bramkamp, M. (2013). Flotillins functionally organize the bacterial membrane. Mol Microbiol 88(6): 1205-1217.