Bradford protein assay

Total protein was harvested from cells lysed in RIPA buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 0.5% sodium deoxycholate, 1% NP-40, 0.1% SDS, 5 mM NaOV4, 10 mM NaF) containing the complete protease inhibitor cocktail (Roche). The concentration of protein was measured using the Bradford protein assay (RotiQuant; Roth) according to the manufacturer’s protocol. 10 μg of total protein from each sample was loaded into 8% or 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) for protein separation. Proteins in the gel were transferred to a polyvinylidene difluoride membrane using the semi-dry transfer cell (Bio-Rad) at 18 V for 45 min. The membranes were blocked by Tris-buffered saline with PBS, 0.1% (vol/vol) Tween-20 (PBST), and 5% (wt/vol) milk powder for 30 min at RT, and then, the membranes were washed with PBST and incubated with anti-SWAP70 at 1 μg/ml (Borggrefe et al, 1998 (link)) O/N at 4°C. Washed by PBST, the polyvinylidene difluoride membrane was incubated with goat anti-rabbit HRP-coupled antibody for 1 h at RT. The protein was detected using enhanced chemiluminescence kits (Millipore Immobilon Western HRP Substrate) and the membrane subsequently developed on Amersham Imager 600 (GE Healthcare). Membranes were subsequently stripped and probed with anti-tubulin as a loading control.

Protein concentrations were quantified using Bradford protein assay (Carl Roth, Germany) following TCA/acetone precipitation. Bovine serum albumin (BSA) was chosen for modeling standard curves and measurements were recorded in triplicates in 96-well plates on EnSpire® Multimode Plate Reader (PerkinElmer, USA). Protein extracts were conveyed on 12% one-dimensional SDS polyacrylamide gel electrophoresis, using Bio-Rad Mini-Protean II Equipment and PageRuler ™ Unstained Protein Ladder (ThermoFischer Scientific, USA), to assess the gross qualitative variances in protein profiles. After electrophoresis, gels were stained with Coomassie brilliant blue (CBB) R-250.

L. monocytogenes (EGD, BUG 600) was cultivated in Luria-Bertani (LB) broth. B. cereus (ATCC 14579) was grown in nutrient broth. For E. faecium (ATCC 6057) columbia agar containing 5% sheep blood (BD BBL, Heidelberg, Germany) or brain-heart-infusion (BHI) medium (Carl Roth, Karlsruhe, Germany) were used. All bacteria strains (Table 1) were cultivated at 37°C. For protein extraction of bacteria grown in the logarithmic and stationary growth phase, bacteria were cultured in broth overnight (stationary phase). The next day, bacterial culture was 1:20 diluted in fresh medium and grown at 37°C to an OD₆₀₀ of approximately 0.8 (logarithmic phase). Bacterial cultures were centrifuged at 4500 × g for 20 min at 4°C. Pelleted bacteria were then harvested in PBS (Sigma, Vienna, Austria) or lysis buffer (20 mM Tris pH 7.5, 100 mM NaCl, 1 % Triton X-100, 0.5 % DOC, 0.1 % SDS, 0.5 % NP-40). Where indicated 5 μg/ml lysozyme (Lactan, Graz, Austria) was added for 1 h at 37°C. Bacteria suspended in PBS or lysis buffer were sonicated on ice 3 times for 45 sec with 50 % power. All bacterial lysates were centrifuged at 16000 × g for 20 min at 4°C. The protein content of the lysates was measured using Bradford protein assay (Carl Roth, Karlsruhe, Germany). All experiments were repeated at least four times.