Peroxidase conjugated goat anti mouse igg

The rCpCDPK2A protein was examined using Western blot analysis. Briefly, the protein was electrophoresed on 10% SDS-PAGE and transferred into a polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, United States). The membrane was probed with anti-His-tag antibodies (Cell Signaling Technology, Danvers, United States) at 1:1,000 dilution as the primary antibody and peroxidase-conjugated goat anti-mouse IgG (Beyotime) at 1:500 dilution as the secondary antibody. The reactivity was visualized using an enhanced High-sig ECL Western Blotting Substrate (Tanon, Shanghai, China).
For Western blot analysis of native CpCDPK2A, 8 × 10⁷ sporozoites of C. parvum were freeze-thawed between −80°C and 4°C in sterile water containing 1% protease inhibitor cocktail (Sigma-Aldrich). The lysates and purified rCpCDPK2A were electrophoresed using 10% SDS-PAGE, transferred into PVDF membranes (Millipore), and incubated with anti-CpCDPK2A polyclonal serum (1:200 dilution) or control serum (1:200 dilution), and incubated with anti-CpCDPK2A monoclonal antibody (1:200 dilution) or purified IgG from native mouse serum (1:200 dilution). The bands in Western blots were developed as described above.

In SDS-PAGE analysis of recombinant EbSWPs, 40 μL protein was added into 10 μL 5×SDS loading buffer and incubated in a water bath at 100°C for 10 min. After polyacrylamide gel electrophoresis of the mixture, the protein in the gel was transferred onto a polyvinylidene fluoride membrane (Millipore). The membrane was blocked with 5% skimmed milk powder for 2 h and reacted with mouse anti-His-tag antibodies (Cell Signaling Technology). Peroxidase-conjugated goat anti-mouse IgG (Beyotime Technology, Shanghai, China) was used as the secondary antibody.