Horseradish peroxidase conjugated anti rabbit igg secondary antibody

Expression and purification of the recombinant CPYs were confirmed by SDS/PAGE and western blotting. The rabbit serum containing polyclonal anti‐CPY antibody (1 : 7500) was prepared in our laboratory. Horseradish peroxidase‐conjugated anti‐rabbit IgG secondary antibody (1 : 100 000) was purchased from GE Healthcare (Buckinghamshire, UK).

For preparation of yeast protein extracts, yeast cells with pYC2-CT vectors encoding BteA protein derivatives (Table S4) were induced for 20 h by cultivation in SC-galactose media. Equivalents of OD₆₀₀ = 1 of yeast cell cultures were collected, and denatured protein extracts were prepared by NaOH lysis/TCA precipitation method, according to (45 (link)). For preparation of mammalian cell protein extracts, HeLa cells at 40% confluency in a 6-well plate were transiently transfected with pEGFP-N2 vectors encoding BteA protein derivatives (Table S4) using Lipofectamine 2000 reagent (Invitrogen). Eighteen hours after transfection, cells were washed with PBS, lysed with 100 μl of ice-cold lysis buffer containing 0.2% Triton X-100 and complete mini protease inhibitors (EDTA free, Roche) in PBS, and clarified by centrifugation (5 min, 10,000g). Extracts were mixed with SDS-PAGE sample loading buffer, heated for 5 min at 50 °C, and separated on 10% SDS-PAGE gels. After the transfer onto nitrocellulose membrane, the proteins were probed overnight with rabbit anti-GFP antibody (1:2000; clone D5.1, Cell Signaling Technology) and revealed by horseradish peroxidase–conjugated anti-rabbit IgG secondary antibody (1:3000; GE Healthcare). Blots were developed using a Pierce ECL chemiluminescence substrate (Thermo Fisher Scientific) and an Image Quant LAS 4000 station (GE Healthcare).

Protein extracts were prepared using Triton X-100 lysis buffer plus Complete Protease Inhibitors (Roche Applied Science) and Protein Phosphatase Inhibitor Cocktail 2 (Sigma-Aldrich). The proteins were separated in NuPAGE 4–12% Bis-Tris gels and transferred to PROTRAN nitrocellulose transfer membranes (Whatman, Springfield Mill, Maidstone, Kent, UK). Membranes were blocked in 3% BSA diluted in TBS-Tween (20mM Tris pH7.6, 137mM NaCl, 0.1% Tween-20). The antibodies were diluted in blocking solution and incubated with the membrane in agitation for 2 hours at room temperature or overnight at 4°C and washed with TBS-Tween before developing with ECL Western Blotting Detection Reagents (GE Healthcare, UK Limited, Little Chalfont, Buckinghamshire, UK) in a FUJIFILM LAS-3000 Intelligent Dark Box (FUJIFILM UK Ltd, Bedford, UK). Antibodies were used at the following concentrations: rabbit anti-Annexin A8 (Eurogentec) 1:1000; rabbit anti-Ki67 (Abcam) 1:1000; goat anti-actin (C-11; Santa Cruz) 1:1000. Anti-goat secondary antibodies conjugated with horseradish peroxidase (DAKO, Glostrup Denmark) were used at 1:2000. Horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody (GE Healthcare) was used at 1:5000.