Tgx tris glycine gels

Samples were separated on 4–20% BioRad TGX Tris-glycine gels and stained overnight with alcian blue as in [40 (link)]. Gels were imaged with a G-box QX4 with white-light converter (Syngene). Band intensity was quantified with GeneQuant (Syngene) via the manual band quantification method, using inter-lane spaces as background. For detection of phosphotyrosines, lysates were blotted onto PVDF membranes, incubated with 4G10 monoclonal antibodies (Millipore; 1:1000 dilution) followed by HRP-conjugated goat anti-mouse (Invitrogen), and developed with ECL-plus substrate (Perkin Elmer). Blots were stripped and re-probed with antiserum raised against Bacillus subtilis isocitrate dehydrogenase (ICDH).

Capsular polysaccharide was extracted from processed cell lysates and analyzed by Alcian blue following described protocols [35 (link)]. Samples were collected from early post-exponential phase cultures (OD 2–3) and centrifuged. Cell pellets were resuspended with lysis buffer [60mM Tris, pH 8; 10mM MgCl₂; 50μM CaCl₂; 20μl/ml DNase (NEB) and RNase cocktail (Amersham); and 3mg/ml lysozyme (Sigma)] and incubated at 37°C for 1 hour. Samples were further processed by 3 liquid nitrogen/37°C freeze-thaw cycles and stepwise treatments with additional DNase and RNase (37°C, 30 minutes), 0.5% SDS (37°C, 30 minutes), and Proteinase K (NEB, 60°C, 1 hour). Samples were combined with SDS-PAGE loading buffer, boiled for 5 minutes, and separated on 4–20% Bio-Rad TGX Tris-glycine gels. Capsule was stained with Alcian blue (Sigma), imaged via white light transillumination (ChemiDoc MP), and quantified using Image Lab (Bio-Rad).