Pierce ecl western blotting chemiluminescence substrate

Female New Zealand white rabbits (Velaz, Czech Republic) were immunized by injection subcutaneously with 100 μg recombinant DbpA or DbpB from B. afzelii A91 in TRIS buffer (1:1) with complete Freund’s adjuvant (Sigma-Aldrich). The rabbit received two boosts of 100 μg recombinant DbpA or DbpB in TRIS buffer (1:1) with incomplete Freund’s adjuvant (Sigma-Aldrich) at 14-day intervals. One week after the final boost, the rabbit was bled to obtain serum. The serum was tested qualitatively by Western blotting to determine the specificity of the antiserum for DbpA and DbpB against recombinant DbpA protein and B. burgdorferi lysates. Briefly, proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose. Membranes were blocked for 2 h with 5% BSA in TRIS-buffered saline with 0.05% Tween 20 (TBS-T). Membranes were washed with TBS-T and incubated for 2 h at room temperature (RT) with DbpA/DbpB antisera diluted 1:200 in blocking buffer. After washing with TBS-T, membranes were incubated for 1 hr at RT with goat anti-rabbit immunoglobulin G conjugated with horseradish peroxidase (HRP) (Vector Laboratories) diluted 1:10,000 in blocking buffer. After a final series of washes with TBS-T, bound antibodies were detected by using Pierce ECL western blotting chemiluminescence substrate (Thermo Scientific).