Irdye 800cw labeled goat anti mouse igg

To further demonstrate the interaction between rTs-Pmy and complement C1q, purified human C1q (5 μg; Merck, Germany) under reducing condition were subjected to SDS-PAGE using 12% polyacrylamide gel followed by either Coomassie bule staining or Far Western blotting. After blocking with 5% dry milk in 1 × PBS, the membrane was incubated with rTs-Pmy (5 μg/ml in PBST and 1% dry milk) at 37°C for 2 h and then washed with PBST. The membrane was probed with an anti-His mAb (rTs-Pmy contains His-tag) (Tiangen, China; 1:5,000 in PBST containing 1% dry milk) for 1 h at room temperature. IRDye 800 CW-labeled goat anti-mouse IgG (LI-COR, Germany; 1:10,000 in PBST containing 1% dry milk) was used as the secondary antibody. Vice versa, rTs-Pmy, non-relevant control BSA and rTs87 (5 μg each) were subjected to 12% SDS-PAGE and then transferred to a NC membrane. After blocking, the membrane was probed with human complement C1q (5 μg/ml in PBST containing 1% dry milk) at 37°C for 2 h. The anti-C1q mAb (Abcam, USA; 1:1,000 in PBST containing 1% dry milk) was used as the primary antibody and IRDye 800 CW-labeled goat anti-mouse IgG (1:10,000 in PBST containing 1% dry milk) was used as the secondary antibody. Membranes were visualized and imaged using an Odyssey infrared imaging system (LI-COR, Germany).

To determine whether the expressed recombinant Ts-Pmy fragments bind to human C9, the fragments and non-relevant control BSA (Sigma, USA) (2 μg each) were subjected to SDS-PAGE under reducing conditions and then transferred to a nitrocellulose membrane. After blocking with 5% milk in PBS, the membrane was incubated with human C9 (Merck, Germany) (1 μg/ml) at 37°C for 2 h and then probed with anti-C9 mAb (0.2 μg/ml; Abnova, Taiwan) at room temperature for 1 h. IRDye 800CW-labeled goat anti-mouse IgG (50 ng/ml; LI-COR, Germany) was used as the secondary antibody.
To determine whether synthesized peptides bind to human C9, the peptides and non-relevant control BSA (Sigma-Aldrich, USA) (5 μg in 2.5 μl each) were spotted onto a nitrocellulose membrane using a narrow-mouth pipette tip. After blocking with 1% BSA in PBS, the membrane was incubated with human C9 (1 μg/ml) at 37°C for 2 h and then with anti-C9 mAb (0.2 μg/ml) at room temperature for 1 h.
All membranes mentioned above were detected and imaged using an Odyssey infrared imaging system (LI-COR, Germany).

To further verify the protein interaction between the human complement C9 and the native Ts-Pmy, immunoprecipitation was performed with T. spiralis adult extracts as described previously [18 (link)]. Protein G MicroBeads (Miltenyi Biotec, Germany) were pre-incubated with 3 μg of human C9 (Merck, Germany) and 2 μg of anti-C9 mAb (IgG1, Abnova, Taiwan) for 30 min on ice. In total, 40 μg of T. spiralis adult extracts was then added and incubated overnight at 4°C with rotation. Beads were washed four times with washing buffers (1% NP40, 50 mM Tris buffer, pH 8.0) before being added with pre-heated 1× SDS gel loading buffer to elute proteins. Samples were separated by SDS-PAGE and probed with anti-Ts-Pmy mAb (7E2) [19 (link)]. IRDye 800CW-labeled goat anti-mouse IgG (LI-COR, Germany) was used as the secondary antibody.