Goat anti rabbit igg hrp conjugate

Full-length WSSV vp11 and the genes of the VP11 interaction candidates were inserted into V5- or FLAG-tagged vectors containing the heat-inducible Drosophila heat shock protein 70 promoter (pDHsp/V5-His and pDHsp/FLAG-His [29] (link)) by PCR cloning, which used WSSV genomic DNA as the template. For DNA transfection, Sf9 insect cells were seeded onto a 6-well plate (8×10⁵ cells/well) and grown overnight. Plasmids containing the appropriate genes (including the empty vector) were transfected into the Sf9 cells and heat shocked and lysed as described above. An aliquot of the supernatant of the lysate (10 µl) was reserved for Western blot analysis to confirm the expression of the transfected genes. The remaining supernatant (90 µl) was then incubated with 15 µl of anti-FLAG M2 affinity gel (Sigma) at 4°C overnight with rotation. The gel was then washed five times in a 150 µl NP-40 lysis buffer. Aliquots of the total cell lysates and immunoprecipitated complexes were separated by 15% SDS-PAGE and transferred to PVDF membrane. V5-tagged fusion proteins were detected with rabbit anti-V5 antibody and goat anti-rabbit IgG-HRP conjugate (Sigma). FLAG-tagged VP11 and VP11 interaction candidate proteins were detected with mouse anti-FLAG monoclonal antibody (Sigma) and goat anti-mouse IgG-HRP conjugate (Sigma). Sequences of the primers used here are listed in Table 2.

A mixture of PA63 and LF were incubated with different amounts of hmPA6 at 4 °C and rotated for 3 h. Next, 50 μL protein-A Sepharose (Invitrogen, USA) was added and incubated at 4 °C. The immune complexes that formed were washed three times with PBST. Subsequently, 50 μL elution buffer was added to separate these antibody-antigen complexes from protein-A Sepharose. As a negative control, LF was incubated with hmPA6 alone. The protein complexes were isolated by running two 10% SDS-PAGE gels; and they were transferred onto a nitrocellulose membrane. The nitrocellulose membranes were blocked at 4 °C overnight, one incubated with 1:5000 diluted rabbit polyclonal anti-PA antibody (Pierce, USA) and the other incubated with anti-LF antibody for 1 h at RT, washed with PBST 3 times, and reacted with 1:4000 diluted goat anti-rabbit IgG-HRP conjugate (Sigma,USA) for an additional 30 min at room temperature. The membrane was washed 3 times with PBST, and the hybridization signal was detected using ECL Western Blot substrate (Millipore, USA). Similar procedures were used for PA21 detection.