Endotoxin free plasmid large scale extraction kit

The selected protein gene C-terminal fusion Myc tag (-Myc) was constructed into the vector pcDNA3.1 (Invitrogen, United States), and the plasmid was extracted using an endotoxin-free plasmid large-scale extraction kit (TIANGEN, China). The pcDNA3.1-BspJ and -Myc plasmids were transfected into HEK293T cells with Lipofectamine 3000 (Invitrogen, United States). After 48 h, IP lysis buffer (Biosharp, China) (containing protein phosphatase inhibitor) was added to harvest the cell proteins. The protein solution was incubated with mouse Anti-6 × His tag antibody and mouse Anti-Myc tag antibody (Abcam, United States) (1:200) at low temperature (4°) for 3 h or overnight, and Protein G-Agarose (Roche, Germany) was added for 4° co-incubation after 3 h or overnight. The precipitate was collected by centrifugation, washed with PBS three times, mixed with SDS-PAGE protein loading solution (CWBIO, China), boiled for five minutes, and subjected to western blot analysis. Mouse Anti-6 × His Tag antibody/mouse Anti-Myc tag antibody (1:1000) was used as the primary antibody; Goat Anti-Mouse IgG H&L (Abcam, United States) (1:2000) was used as the secondary antibody, and a SuperSignal^TM West Femto Trial Kit (Thermo Fisher Scientific, United States) was used for color developing.

We searched the BspJ gene sequence in the Genbank database. Primer 5.0 was employed to design the primers used to amplify the target fragment (BspJ-F 5′-ATGAAGAGCTTGCAGTTTTC-3′, BspJ-R 5′-TTATCGATATGCCCGAGGTAC-3′). Brucella abortus was used to as a genome template for BspJ gene cloning. The C-terminal fusion His tag of the BspJ gene constructed to pDsRed2-C1 (Clontech, United States) and pcDNA3.1 (Invitrogen, United States) vectors. Using an endotoxin-free plasmid large-scale extraction kit (TIANGEN, China) to extract the plasmids pDsRed2-C1-BspJ and pcDNA3.1-BspJ. Meanwhile, the BspJ gene was constructed into the vector pGBKT7 (Clontech, United States).