Anti-NDP52 抗体 (ab68588)

Western blot: Anti-CALCOCO2 antibody (ab68588) staining at 1 ug/ml, shown in green; Mouse anti-CANX [CANX/1543] (ab238078) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab68588 was shown to bind specifically to CALCOCO2. A band was observed at 57 kDa in wild-type A549 cell lysates with no signal observed at this size in CALCOCO2 knockout cell line. To generate this image, wild-type and CALCOCO2 knockout A549 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

ab68588 staining NDP52 in HeLa cells +/- Chloroquine (50μM, 24 hours). The cells were fixed with 4% paraformaldheyde (10 min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab68588 at 5μg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control, at 1/1000 dilution. The secondary antibodies were  ab150081 (colored green) and ab150120 (pseudo-colored red) used at 1/1000 dilution for 1 hour at room temperature. DAPI was used to stain the cell nuclei (colored blue) at a concentration of 1.43 µM for 1 hour at room temperature.

IHC image of NDP52 staining in Human Cerebral Cortex FFPE section, performed on a BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab68588, 5µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX