Uvp chemstudio plus imager

The upstream regions of the toxP, cbuA0029, and groEL genes were first selected for PCR amplification. PCR products (1 μg) of desired templates were 3′ end‐labeled using a Pierce biotin 3′ End DNA Labeling Kit (Thermo Scientific). The resulting probe reaction mixtures were electrophoresed on a 0.8% agarose gel for 30 min at 100 V and then gel purified with a NucleoSpin Gel and PCR Clean‐up kit (Takara Bio USA). The EMSA binding reaction, consisting of 2.5% glycerol, 5 mM MgCl2, 50 mM KCl, 1 nM biotin‐labeled DNA, and varying concentrations of either ToxP or AntitoxP/ToxP in 1X Binding Buffer (LightShift Chemiluminescence kit; Thermo Scientific), was assembled and incubated at room temperature for 30 min. A nondenaturing loading dye (0.25% bromophenol blue) was added, and the resulting RNA mixtures were resolved on a 10% polyacrylamide gel for 2 h at 100 V. DNA/protein complexes were transferred to a Hybond‐N+ positively charged nylon membrane (Amersham Pharmacia Biotech) using an electroblot transfer system (Bio‐Rad) and cross‐linked with short‐wave UV light in a GS gene linker UV chamber (Bio‐Rad). A North2South chemiluminescence hybridization and detection kit (Thermo Scientific) was used to detect resulting bands. The blot was imaged on a UVP ChemStudio PLUS Imager (Analytik Jena).

C. burnetii production of IcmD, IcmK, dCas9‐3x‐cMyc, GST‐AntitoxP, ToxP‐V5‐6xHis, and XpressT‐6xHis‐CBU0665 was examined by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and immunoblotting. PVDF membrane was incubated with the following antibodies: polyclonal rabbit anti‐IcmD antibody (1:5000), polyclonal rabbit anti‐IcmK antibody (1:2500; generously provided by Edward Shaw, PCOM South Georgia), mouse monoclonal anti‐c‐myc antibody (1:5000; clone 9E10 BD Biosciences), goat polyclonal anti‐GST antibody (1:5000, Sigma‐Aldrich), mouse monoclonal anti‐V5 antibody (1:5000; Invitrogen), and mouse monoclonal anti‐XpressT antibody (1:5000; Invitrogen). Following incubation of membranes with primary antibody, reacting proteins were detected using anti‐rabbit (IcmD and IcmK), antigoat (GST‐AntitoxP), or antimouse (dCas9‐3x‐c‐myc, ToxP‐V5‐6xHis, and XpressT‐6xHis‐CBU0665) IgG secondary antibodies conjugated to horseradish peroxidase (Pierce, Rockford, IL) and chemiluminescence using ECL Pico or Femto reagent (Pierce). Chemiluminescence was detected using the UVP ChemStudio plus imager (Analytik Jena), and images were processed using the VisionWorks software (Version 9.1.21054.7804).