HRP Anti-Caveolin-1 antibody [E249] - Caveolae Marker

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - HRP Anti-Caveolin-1 antibody [E249] - Caveolae Marker (AB193893)

IHC image of Caveolin-1 staining in a section of formalin-fixed paraffin-embedded human normal lung*. The section was pre-treated using pressure cooker heat mediated antigen retrieval with sodium citrate buffer (pH6) for 30mins, and incubated overnight at +4°C with ab193893 at a working dilution of 1/500. DAB was used as the chromogen (ab103723), diluted 1/100 and incubated for 10min at room temperature. The section was counterstained with haematoxylin and mounted with DPX. The inset negative control image is taken from an identical assay without primary antibody.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

• WB

Lab

Western blot - HRP Anti-Caveolin-1 antibody [E249] - Caveolae Marker (AB193893)

Lanes 1 - 4 : Merged signal (red and green). Green - ab193893 observed at 20 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37 kDa.

ab193893 was shown to react with Caveolin-1 in wild-type HeLa cells in Western blot with loss of signal observed in CAV1 knockout cell line ab255371 (CAV1 knockout cell lysate ab263806). Wild-type HeLa and CAV1 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween^®) before incubation with ab193893 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4 °C at a 1 in 5000 dilution and a 1 in 20000 dilution respectively. Blots were incubated with and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 h at room temperature. Blots were developed with Optiblot ECL reagent (ab133456) before imaging.

All lanes:

Western blot - HRP Anti-Caveolin-1 antibody [E249] - Caveolae Marker (ab193893) at 1/5000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

CAV1 knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human CAV1 (Caveolin-1) knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-cav1-caveolin-1-knockout-hela-cell-line-ab255371'>ab255371</a>)

Lane 3:

A431 cell lysate at 20 µg

Lane 4:

A549 cell lysate at 20 µg

Predicted band size: 20 kDa

Observed band size: 20 kDa

false

Exposure time: 90s

• WB

Lab

Western blot - HRP Anti-Caveolin-1 antibody [E249] - Caveolae Marker (AB193893)

This blot was produced using a 4-12% Bis-tris gel under the MES buffer system. The gel was run at 200V for 35 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 2% Bovine Serum Albumin before being incubated with ab193893 overnight at 4°C. Antibody binding was visualised using ECL development solution ab133406.

All lanes:

Western blot - HRP Anti-Caveolin-1 antibody [E249] - Caveolae Marker (ab193893) at 1/5000 dilution

All lanes:

A431 (Human epithelial carcinoma cell line) Whole Cell Lysate at 10 µg

Predicted band size: 20 kDa

Observed band size: 17 kDa,20 kDa

true

Exposure time: 10s