Detection of Spo0A phosphorylation using a Phos-tag acrylamide gel

Phos-tag acrylamide gels were prepared according to the instructions provided (Wako); 10% acrylamide gels were copolymerized with 25 nM Phos-tag acrylamide and 10 nM MnCl2. 20-ml of bacterial cultures were centrifuged at 5,000 g at 4 °C for 10 min, and the pellets suspended in 1 ml 10 mM Tris pH 8.0. The cells were lysed using a French pressure cell (18,000 lb/in2). Samples were stored on ice prior to loading onto Phos-tag acrylamide gels and run at 4 °C. Gels were fixed for 10 min in transfer buffer with 10 mM EDTA and then washed for 10 min in transfer buffer without EDTA twice. After transfer to a nitrocellulose membrane, the samples were probed with rabbit polyclonal anti-Spo0A or anti-FliC antibodies at a 1:1000 dilution and an anti-rabbit secondary antibody conjugated to horseradish peroxidase (Sigma) was used at dilution 1:10,000. Images were adjusted and cropped and quantified using ImageJ (http://rsbweb.nih.gov/ij/). In order to dephosphorylate Spo0A ~ P, samples were incubated at 100 °C for 5 min.