Alexa fluor 680 igg antibody

Bacterial pellets were resuspended in 50 mM Tris-HCl, pH 8.0, with protease inhibitors and lysed at 10, 000 PSI using a cell disruptor (Constant Systems, Kennesaw, GA). Cell debris was pelleted by centrifugation at 15,000 × g for 15 min at 4 °C, and the clear supernatant was centrifuged at 40,000 × g for 30 min at 4 °C. The pellet, containing total membranes, was suspended 50 mM Tris-HCl, pH 8.0. Protein concentration was determined by the Bradford assay (Bio-Rad). Proteins were separated by SDS-PAGE on a 12% or 18% gel (if the experiment included detection of HA-SoxY) and transferred to a nitrocellulose membrane. Immunoblots were probed with anti-FLAG and anti-HA (Sigma) mouse monoclonal antibodies. Secondary anti- mouse Alexa fluor-680 IgG antibody (Invitrogen) was used to detect fluorescence using an Odyssey infrared imaging system (LI-COR Biosciences).

Overnight cultures were diluted to an optical density at 600 nm (OD₆₀₀) of 0.03 in 30 ml of fresh LB medium with or without PmB and incubated for 3.5 h at 37°C at 200 rpm. Following incubation, cells equivalent to an OD₆₀₀ of ~0.2 were pelleted, resuspended in 30 µl of SDS-PAGE protein loading dye, and boiled to obtain whole-cell lysates. Secreted proteins were precipitated from the supernatant of the rest of the cultures with 10% trichloroacetic acid as previously described (51 (link)). Precipitated proteins were resuspended in Tris buffer at 1 M and pH 7.5. The volume of protein samples loaded onto the 16% SDS-polyacrylamide gel was normalized to the OD₆₀₀ value. After SDS-PAGE, proteins were transferred onto nitrocellulose membranes and the membranes were blocked overnight at 4°C with Western blocking reagent (Roche Diagnostics, Laval, QC, Canada) in TBST (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.1% Tween 20). The primary antibody, anti-FLAG M2 monoclonal antibody (Sigma) or anti-α-subunit RNA polymerase (E. coli) (Neoclone, Madison, WI), was diluted to 1:15,000 in TBST and applied for 1.5 h. The secondary antibody, goat anti-mouse Alexa Fluor 680 IgG antibody (Invitrogen), was diluted to 1:15,000 and applied for 1 h. Western blot assays were developed with the LI-COR Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE).