Anti mouse igg hrp linked secondary antibody

The antibody response in mice was determined by endpoint titer ELISA using a protocol similar to that described previously [18 (link)]. Briefly, 96-well plates (Costar, Corning, NY) were coated with 2 μg/ml of MBP-OspA diluted in PBS and incubated overnight at 4°C. The MBP-OspA was removed and the plates were blocked with 1% BSA dissolved in PBS. After removing the blocking reagent, the top well of each lane was loaded in duplicate with serum dilutions. The samples were serially diluted in equal volume of PBS. Following overnight incubation at 4°C, the samples were aspirated and the plates were washed 0.1% Tween 20 in PBS. The plates were incubated with anti-mouse IgG, HRP-linked secondary antibody (1:10,000 dilution in PBS) (Cell Signaling Technology) for 1 h at room temperature. The secondary antibody was aspirated, washed and incubated with Sure Blue TMB peroxidase substrate (KPL, Gaithersburg, MD) followed by addition of equal volume of stop solution (1N HCl solution). Absorbance was measured at 450 nm. Serum from unvaccinated mice was used as the negative control. The cutoff value was determined as three standard deviations above the average OD of negative controls. The reciprocal of highest dilution above the cut-off was defined as endpoint antibody titer of each serum sample.

Laemmli buffer containing 5% β-ME was added to samples, boiled for 5 min, and analyzed on NuPage 4–12% Bis-Tris polyacrylamide gels (Life Technologies). Following transfer to PVDF membranes, blots were blocked in 20 mM Tris-buffered saline, pH 7.6, containing 0.1% Tween 20 (TBST) and 5% (wt/vol) non-fat dry milk for 30 minutes at room temperature. Blots were subsequently incubated overnight at 4°C with primary anti-FLAG antibody (#F3165, MilliporeSigma) at 1:2,000 (v/v), followed by a 4-hour incubation at room temperature in anti-mouse IgG, HRP-linked secondary antibody (#7076, Cell Signaling) at 1:4,000 (v/v).