Clarity western ecl substrate

To monitor the expression patterns of the DEAD-box RNA helicases, we used strains expressing the helicases labeled with a C-terminal triple FLAG tag. The respective strains for CshA, CshB, DeaD, and YfmL were available (Lehnik-Habrink et al., 2013 (link)). Single mutants were transformed with chromosomal DNA thus replacing the respective genes with a Flag- tagged construct.
LB media cultures were grown at 18 and 37°C until the exponential phase. Cells were harvested by centrifugation (950 × g, 10 min, 25°C) and the pellet washed (with 5 mL Tris 50 mM, pH 8.4, KCl 1 M) and lysed with a buffer (Tris 50 mM, pH 8.4, KCl 1 M) containing 1X protease inhibitor (Thermo Fisher), 1X DNase (Sigma-Aldrich), 50 μg/ml lysozyme (USB^®) and centrifuged (12,000 × g 15 min at 4°C). The aqueous phase was separated (Cutting and Vander Horn, 1990 ). The protein quantification was determinated using Bio-Rad’s Bradford reagent.
For Western blot analysis, proteins were separated on a 12% polyacrylamide gel and transferred onto nitrocellulose membrane (Bio-Rad) by electroblotting. Rabbit anti-FLAG polyclonal antibodies (Sigma-Aldrich; 1X) served as primary antibody. The antibodies were visualized by using 100 μl Clarity Western ECL Substrate (Peroxide solution) and 100 μl Clarity Western ECL Substrate (Luminol/enhancer solution) from Bio-Rad (Lehnik-Habrink et al., 2010 (link)).

SDS-PAGE and immunoblotting was performend according to standard procedures^{67 (link)}. Briefly, samples of bacterial cultures were harvested by centrifugation at 10,000×g to separate cells from the culture supernatant. Cell pellets were suspended in appropriate volume of 1 × Laemmli buffer and boiled to obtain whole cell lysates. Residual culture supernatants were filtered through 0.45 µm PVDF syringe filters (Millipore, USA) and subjected to protein precipitation with 10% trichloroacetic acid (TCA). Precipitated proteins were collected by centrifugation at 15,000×g for 15 min at 4 °C and dissolved by boiling for 5 min in 1 × Laemmli buffer. Protein samples were resolved on a SDS-PAGE and processed for immunoblotting. HRP-conjugated donkey anti-rabbit IgG (Agrisera AB, Sweden) was used as a secondary antibody. Detection was performed using Clarity® western ECL substrate (Bio Rad). Pre-stained protein molecular weight standards (SM0679, Fermentas) were used to determine the protein sizes. HRP-conjugated donkey anti-rabbit IgG 1:10,000 dilution (Agrisera AB) was used as a secondary antibody. Detection was performed using Clarity® western ECL substrate (Bio Rad). Protein molecular weight standards (SM0679, Fermentas) were used to determine the protein sizes.