Anti mouse igg horseradish peroxidase conjugated secondary antibody

Protein was extracted from cell pellets using ice-cold RIPA buffer with protease and phosphatase inhibitors (Roche Diagnostics Ltd., Sussex, UK), and protein concentration calculated using the bicinchoninic acid assay. Twenty micrograms of protein were separated using Novex^® 4–12% Tris–Glycine gels and transferred to a nitrocellulose membrane by semi-dry transfer. Membranes were blocked with 5% (w/v) BSA in Tris-buffered saline containing 0.05% (v/v) Tween-20 (TBST), and membranes were then incubated with antibodies directed to DAPK1 (clone55; 1:250 dilution; Sigma Aldrich, Poole, UK) or phosphorylated DAPK1 (pSer308; 1 µg/mL dilution; Novus Biologicals, Cambridge, UK) overnight at 4 °C or with anti-β-actin (1:30,000; Cell Signalling Technologies^®, Danvers, MA) for 1 h at room temperature. Membranes were washed and incubated with anti-mouse IgG horseradish peroxidase-conjugated secondary antibody (1:3000 dilution; Cell Signalling Technologies^®). All antibodies were diluted in 5% BSA/TBST. Immuno-reactive proteins were visualized using an enhanced chemiluminescent substrate for detection of horseradish peroxidase activity (Pierce^™ ECL Western Blotting Substrate, ThermoFisher).

S. sanguinis cells were grown overnight in TH medium and washed twice with PBS, then resuspended in protoplasting buffer containing 0.1 M KPO₄, pH 6.2, 0.3 M raffinose, 10 mM MgCl₂, complete EDTA-free protease inhibitors (Roche, Switzerland) and 200 units/ml of mutanolysin (Sigma Aldrich). The cells were incubated at 37°C for 3 h with mild rotation. Protoplasts were sedimented by centrifugation at 20,000×g for 20 min. Supernatants were collected as cell wall fractions, and proteins in the fractions were separated by SDS-PAGE and blotted onto polyvinylidene difluoride membranes (Millipore). Each membrane was blocked with a casein-based solution (Megmilk Snow Brand, Japan) and incubated with mouse anti-SWAN antiserum (1∶2000) for 1 h at room temperature, followed by washing steps and incubation with an anti-mouse IgG horseradish peroxidase-conjugated secondary antibody (Cell Signaling Technology, MA, USA). The membranes were then washed and developed with a Western blotting substrate (Pierce).