Immunoblotting Analysis of Bacterial Chemotaxis Proteins

Cells were pelleted by centrifugation (6000 x g) and resuspended at an OD₆₀₀ of 2 in 10 mM potassium phosphate (pH 7.0) and 0.1 mM EDTA. Cells from 0.5 ml of the suspension were pelleted and lysed by boiling in 50 μl of sample buffer (Laemmli, 1970 (link)). Proteins released from the lysed cells were analysed by electrophoresis in sodium dodecyl sulphate-containing polyacrylamide gels (SDS-PAGE) and visualized by immunoblotting with an antiserum directed against the highly conserved portion of the Tsr signalling domain (Ames and Parkinson, 1994 (link)) in 10% acrylamide, 0.05% bisacrylamide gels (Studdert and Parkinson, 2004 (link)), or an antiserum against CheW in 17.5% acrylamide, 0.47% bisacrylamide gels (Cardozo et al., 2010 (link)). Either Cy5-labeled (Amersham) or alkaline phosphatase-conjugated (Sigma) goat anti-rabbit immunoglobulin were used as secondary antibodies. Cy5-labeled antibodies were detected with a Storm 840 fluorimager (Amersham); alkaline phosphatase-conjugated antibodies were developed with nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate (both from Promega) and converted to grey scale images with a digital scanner. All gel images were analysed with ImageQuant (Amersham).