Any kd tgx sds page gels

Isogenic V. cholerae strains either lacking PLE or with an integrated PLE (PLE1 to -5) were grown to an OD₆₀₀ of 0.3 and infected with ICP1_2006E ΔCRISPR Δcas2-3 at an MOI of 1 and returned to the incubator at 37°C with aeration. At 16 min after phage addition, 1 ml of infected culture was collected and mixed with an equal volume of ice-cold methanol. Samples were centrifuged at 5,000 × g for 10 min at 4°C to pellet infected cells. Pellets were washed once with ice-cold PBS and resuspended in lysis buffer (50 mM Tris, 150 mM NaCl, 1 mM EDTA, 0.5% Triton X-100, 1× protease inhibitor [Thermo Pierce Protease and Phosphatase inhibitor tablet]). Protein concentration was quantified with a Pierce bicinchoninic acid (BCA) protein assay kit (Thermo). Thirty micrograms of total protein sample was mixed with Laemmli buffer (Bio-Rad) and boiled at 99°C for 10 min. Samples were run on Any-kD TGX-SDS-PAGE gels (Bio-Rad) and transferred to nitrocellulose membranes with a Transblot Turbo Transfer system (Bio-Rad). Custom primary peptide antibody generated in rabbits against ICP1 capsid (Gp122, YP_004251064.1) (GenScript) was diluted 1:1,500. Band detection was conducted with a goat anti-rabbit IgG horseradish peroxidase (HRP) secondary antibody (Bio-Rad) at 1:10,000 followed by development with Clarity Western ECL substrate (Bio-Rad) and imaging on a ChemiDoc XRS imaging system (Bio-Rad).