Rabbit anti 6 his tag antibody

Expression levels of MdrP or its variants were judged by Western blot using the everted membrane vesicles from E. coli CM2 transformants. Everted membrane vesicles with 10 μg proteins were subjected to 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted to nitrocellulose membranes. A rabbit anti-6 × His-tag antibody (Abcam Ltd., China) and a goat antirabbit secondary antibody (Nachuan Biotechnology Co., Ltd., Changchun, China) were used. Positive signals were probed by a horseradish peroxidase (Nachuan Biotechnology Co., Ltd., Changchun, China) via a Tanon-5200 multiautomatic chemiluminescence/fluorescence imaging system (Tanon Co., Ltd., China).

For western blotting (WB), the proteins were resolved on SDS–polyacrylamide gel electrophoresis (SDS–PAGE) gels followed by standard WB. Rabbit anti-6×His tag antibody (Abcam, Cambridge, MA, USA) (1:2,000) as the primary antibody. Goat anti-rabbit IgG H&L (Abcam, Cambridge, MA, USA) (1:4,000) was used as the secondary antibody and a SuperSignal West Femto Trial Kit (Thermo Fisher Scientific, Waltham, MA, USA) was used for color development. β-Actin protein was used as the control, using mouse anti-β-actin monoclonal antibody (Sino Biological, Beijing, China) (1:2,000) and goat anti-mouse IgG H&L (Abcam, Cambridge, MA, USA) (1:4,000).

HEK293T cells were cultured in a cell culture plate at an order of magnitude of 1 × 10⁷ cells/well. When the cell density reached 80∼90%, Lipofectamine 3000 (Invitrogen, United States) was used to transfect the plasmid (pcDNA3.1-BspJ) into the HEK293T cell. After 48 h, a nuclear protein extraction kit (BestBio, China) was employed to extract the nuclear and cytoplasmic proteins. This was followed by SDS-PAGE (CWBIO, China) for western blot analysis using rabbit anti-6 × His tag antibody (Abcam, United States) (1:2000) as the primary antibody. Goat anti rabbit IgG H&L (Abcam, United States) (1:4000) was used as the secondary antibody, and a SuperSignal^TM West Femto Trial kit (Thermo Fisher Scientific, United States) was used for color developing. Nucleoprotein histone was used as control. The primary antibody Mouse Anti-Histone H3 Monoclonal Antibody (SinoBiological, China) (1:2000) and the secondary antibody Goat Anti-Mouse IgG H&L (Abcam, United States) (1: 4000) were used in the experiment.