Ab200537

Protein fractions were analyzed by standard denaturing SDS-PAGE using 4% to 20% gradient mini-gels (Bio-Rad), Expedeon InstantBlue Coomassie stain, and a Li-Cor Odyssey Fc system for Coomassie visualization (700-nm channel). For Western blot analyses, SDS-PAGE-migrated proteins were directly transferred using a standard mini-gel transfer protocol, 0.2-μm polyvinylidene difluoride (PVDF) membranes, and a Trans-blot Turbo transfer system (Bio-Rad). Blocking and antibody incubations were in the presence of 5% skim milk in Tris-phosphate-buffered saline (TPBS); all washes between and after antibody incubations were with 1× TPBS buffer. Rabbit anti-His₆ (dilution 1:1,000, ab200537; Abcam) and mouse anti-HA (dilution 1:1,000, number 26183; Thermo Fisher Scientific) antibodies were used as primary antibodies; Alexa Fluor 680-conjugated goat ant-rabbit (dilution 1:10,000, ab175773; Abcam) and donkey anti-mouse (dilution 1:10,000, ab175774; Abcam) were used as secondary antibodies. The Alexa Fluor 680 signal was detected using a Li-Cor Odyssey Fc system in the 700-nm channel.

Total protein samples corresponding to 1 OD₆₀₀ units were collected at the desired OD₆₀₀ and cell pellets were resuspended in 1× Laemmli buffer to a final concentration of 0.01 OD per µl. The samples were immunoblotted as previously described (Papenfort et al, 2015 (link)). Signals were visualized using a Fusion FX EDGE imager and band intensities were quantified using the BIO‐1D software (both from Vilber Lourmat, Marne‐la‐Vallée, France). 3xFlag and SPA‐tagged proteins were detected using mouse anti‐Flag antibody (#F1804, Sigma‐Aldrich, St. Louis, Missouri). The SPA epitope contains the 3×FLAG and the calmodulin‐binding peptide sequences separated by a TEV protease cleavage site (Zeghouf et al, 2004 (link)). HA and 6xHis‐tagged samples were detected using mouse monoclonal anti‐HA (#ab18181, Abcam, Cambridge, UK) and rabbit monoclonal anti‐6xHis (#ab200537, Abcam, Cambridge, UK) antibodies, respectively. RNAP served as a loading control and was detected using rabbit anti‐RNAP antibody (#WP003, BioLegend, San Diego, California). The corresponding secondary antibodies used were goat anti‐mouse HRP‐conjugated IgG antibody, (#31430, Thermo Fisher Scientific, Waltham, Massachusetts) and goat anti‐rabbit HRP‐conjugated IgG antibody, (#16104, Thermo Fisher Scientific, Waltham, Massachusetts).