Materials and Reagents

Bacillus subtilis (B. subtilis)
Glycerol (Carl Roth, catalog number: 7530.4 )
Tris (J.T.Baker^®, catalog number: 1414 )
NaCl (AppliChem GmbH, catalog number: A3597.5000 )
MgCl₂ (Merck KGaA, catalog number: 1.05833.1000 )
Proteinase inhibitor (Roche Diagnostics, catalog number: 0 4693159001 )
DNAse I (Roche Diagnostics, catalog number: 10104159001 )
Lysozyme (Roche Diagnostics, catalog number: 10153516103 )
Casein hydrolysate (Oxoid Limited, catalog number: LP0041 )
Buffer A (see Recipes)
Casein Hydrolysate (CH-medium) (see Recipes)
Solution G (see Recipes)

Equipment

Glass beads (diameter 0.2-0.3 mm) (Sigma-Aldrich, catalog number: G1277 )
French press homogenizer (Glen Mills, French press G-M^TM )
Ultra-centrifuge (Beckman Coulter, model: optima^TM XPN-100 )
Ti-70 Rotor (Beckman Coulter)
FastPrep tissue homogenizer (MP Biomedicals, model: 116004500 )
Refrigerated centrifuge (e.g. Beckman Coulter, model: Avanti-J25 )
Acrodisc^® syringe filters (a pore size of 0.2 µm) (Pall, catalog number: 4652 )

Procedure

Inoculate B. subtilis to an OD₆₀₀ of 0.1 in appropriate medium [e.g. CH, lysogeny broth (LB), Spizizen minimal medium (SMM); here we used 50 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 250 ml flasks with baffles).
Harvest cells by centrifugation (10,000 x g; 15 min; 4 °C).
Discard supernatant and resuspend cells in 1/5 volumes of original culture at 4 °C in pre-cooled Buffer A.
Centrifuge again (10,000 x g; 15 min; 4 °C).
Discard supernatant and resuspend cells in 5-8x times volume of the cell pellet in Buffer A at 4 °C supplemented with proteinase inhibitor and DNase I. Use concentrations as specified by the manufacturer.
Disrupt cells
Note: If cell disruption is critical, the cell suspension can be incubated with lysozyme (50 μg/ml) on ice for 30-120 min prior to cell disruption, check microscopically.
1. Small cell volumes (1 ml in reaction tubes) can be disrupted in a FastPrep tissue homogenizer for 30 sec at 6.5 m/s with diameter 0.2-0.3 mm glass beads and cooling for 5 min on ice between runs (minimum 5 runs).
2. Larger cell volumes can be disrupted in a French press homogenizer at 125 MPa and 3-5 passes. Cool sample on ice for 5 min between every pass. Note that only use of a French Press system will yield inside-out vesicles.
Remove cell debris by centrifugation (12,000 x g; 15 min; 4 °C).
Collect the supernatant (cell lysate) and centrifuge it at ≥ 200,000 x g; 4 °C and 60-90 min (e.g. in a Beckman-Coulter^® Ti-70 rotor at 45,000 rpm).
Note: Longer centrifugation time results in a better separation of membranes at high protein concentrations.
The supernatant represents the cytoplasmatic fraction; the pellet contains B. subtilis membranes (lipids, trans-membrane proteins and membrane associated proteins).
Note: Resuspend the pellet in an appropriate buffer, e.g. Buffer A.

Recipes

Buffer A
50 mM Tris.HCl (pH 7.5)
150 mM NaCl
5 mM MgCl₂
10% Glycerol (v/v)
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 x ml^-1 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

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.