Novex 4 20 tris glycine polyacrylamide gels

Equivalent amounts of B. burgdorferi whole cell extracts were analyzed by SDS-PAGE using Novex 4–20% Tris-Glycine polyacrylamide gels (Invitrogen) and proteins transferred to PVDF Immobilon membranes (Millipore). Membranes were blocked overnight at 4°C in blocking buffer (also used to dilute antibodies), which consisted of dPBS + 0.5% Tween-20 + 4% dried milk + 1% goat serum. Membranes were probed with rabbit anti-OspC (1:1000), mouse anti-OspA antibodies (1,2500, CDC), mouse anti-FlaB (1,100, gift from Tom Schwan), rabbit anti-GpsA (1:1000) or rabbit anti-GlpD (1,1000) followed by goat anti-rabbit or goat anti-mouse HRP-linked antibodies (Bio-Rad Laboratories) (1,10,000). Anti-GpsA and anti-GlpD antibodies were produced by GenScript using B. hermsii peptides as antigens. Detection was done by chemiluminescence (Amersham ECL Prime, GE Healthcare) and visualized using an LAS-3000 Intelligent Dark Box (Fujifilm Medical Systems USA).

Desalting and buffer exchange of all antigens was performed using Micro Spin columns (PD Mini Trap-G25, GE Healthcare) according to the manufacturer’s protocol. The quality of the antigens was assessed by Western blot and Silver stain (Fluka). Briefly, following mixing in an equal volume of 2X Laemmli sample buffer and heating at 90°C for 5min, similar quantities of TAAs (1 ug) were separated on Novex 4–20% Tris glycine polyacrylamide gels (Invitrogen Life Technologies, USA). After transfer onto nitrocellulose, antigens were detected by labelling with rabbit anti-His antibodies (1:1,000) followed by a horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibody (1:2,000). Blocking and incubations with antibodies were performed with 0.5% milk in Tris-buffered saline, whereas 0.05% Tween-20 in Tris-buffered saline was used for washing. The Amersham ECL Prime Western Blotting Detection Reagent was used to reveal HRP activity according to the manufacturer’s instructions.