Hrp conjugated goat anti mouse ab

To quantify NY‐ESO‐1 protein levels by western blot analysis, 2 × 10⁶ aAVC‐NY‐ESO‐1 cells were lysed in 150 μL Laemmli sample buffer (Bio‐Rad). Samples were boiled at 95°C for 5 minutes, and 15 μL of samples, as well as molecular weight marker (Bio‐Rad), were loaded and separated on a polyacrylamide gel. Protein was then electrotransferred onto a PVDF membrane. The membranes were blocked in TBS‐T containing 5% skim milk and then incubated with the primary anti‐NY‐ESO‐1 Ab (clone, E978; Santa Cruz Biotechnology, #sc‐53869) at 4°C with gentle shaking overnight. The membranes were washed with TBS‐T for 10 minutes three times and incubated with HRP‐conjugated goat anti‐mouse Ab (R&D Systems) at RT with gentle shaking for 1 hour. Chemiluminescence images were acquired using a luminescence image analyzer (LAS 1000) and Image Gauge software (Fujifilm Co.). Following chemiluminescence imaging, a bright‐field image of the PVDF membrane was obtained. Protein levels were measured using Image Gauge software, and image processing (resizing, cropping, and merging) was undertaken using Adobe Photoshop (Adobe Systems).