Anti gst

TYLCV V2, SlGRXC6, and SlNTRC80 ORFs were cloned into pET32a, pGEX4T-1, or pHMTC to make constructs expressing His6-, GST-, or MBP-tagged proteins. All constructs were transformed into E. coli BL21 (DE3) cells and cultured at 37°C. After the OD₆₀₀ had reached ∼0.6, β-D-thiogalactoside (IPTG) was added to the cultures and incubated for 4 hours. Bacterial cells were pelleted, resuspended with phosphate-buffered saline (PBS) buffer, and sonicated to break cells. The His6-, GST-, and MBP-fused proteins were individually purified using a nickel-nitrilotriacetic acid (Ni-NTA) resin binding column (Qiagen, Germany), GST binding column (Qiagen, Germany), and MBP binding column (Novagen, Germany), respectively, according to manufacturer’s instructions. Competitive pulldown assays were performed as described previously [20 (link)] with minor modifications. Briefly, the indicated amounts of His6-V2 or His6 were mixed with 2 μg of GST-SlGRXC6 for 1 h before being incubated with 2 μg of MBP-SlNTRC80 for pulldown assays. The column-bound proteins were eluted and detected by immunoblotting with anti-His6 (Abcam, UK), anti-MBP (Sigma, USA), or anti-GST (GenScript, USA) antibody.