Immobilon p membrane filter

Cells were grown at 30 °C in M9-based medium with 20 μg/ml each of the 20 amino acids, 2 μg/ml thiamine, and 0.4% glucose until mid-log phase. Total cellular proteins were precipitated with 5% trichloroacetic acid, washed with acetone, and dissolved in SDS sample buffer. Immunoblotting was carried out essentially as described previously (90 (link), 91 (link)). Proteins were separated by SDS-PAGE and electroblotted onto an Immobilon-P membrane filter (MilliporeSigma). Only when 15% bis-Tris gel was used for SDS-PAGE, a transferred membrane filter was dried at 37 °C for 30 min and then hydrophilized with methanol. After blocking with BLOTTO (90 (link)), the filter was incubated with an appropriate antibody. For anti-RseP immunoblotting, anti-RseP antibodies were preincubated with whole-cell lysates of AD1840 (the ΔrseA ΔrseP ΔdegS strain) at 4 °C for 1 h to reduce a background as described previously (82 (link)). The filter was then washed and incubated with goat anti-mouse or anti-rabbit IgG conjugated with horseradish peroxide (Bio-Rad). After washing of the filter, proteins that reacted with secondary antibodies were visualized using ECL or ECL Prime Western Blotting Detection Reagents (Cytiva) and Bio image analyzer LAS4000mini (Cytiva).