All lanes : Anti-Caveolin-1 antibody [E249] - Caveolae Marker (ab32577) at 1/1000 dilution

Lane 1 : Wild-type A431 cell lysate
Lane 2 : CAV1 knockout A431 cell lysate
Lane 3 : A549 cell lysate
Lane 4 : HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 20 kDa
Observed band size: 20 kDa

Lanes 1 - 4: Merged signal (red and green). Green - ab32577 observed at 20 kDa. Red - loading control, ab7291 (Mouse anti-Alpha Tubulin [DM1A] observed at 55kDa.

ab32577 was shown to react with Caveolin-1 in wild-type A431 cells in western blot. Loss of signal was observed when CAV1 knockout sample was used. A431 wild-type 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 ab32577 and ab7291 (Mouse anti-Alpha Tubulin [DM1A] overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

ab32577 staining Caveolin-1 in wild-type HeLa cells (top panel) and CAV1 knockout HeLa cells (ab255371) (bottom panel). The cells were fixed with 100% methanol (5 min) then 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 with ab32577 at 1/200 dilution and ab7291 (Mouse monoclonal to alpha Tubulin) at 1/1000 dilution overnight at 4°C followed by a further incubation at room temperature for 1h with a goat secondary antibody to rabbit IgG (Alexa Fluor^® 488) (ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to mouse IgG (Alexa Fluor^® 594) (ab150120) at 2 μg/ml (shown in red). Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems TCS SP8).

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Rat colon tissue sections labeling Caveolin-1 with Purified ab32577 at 1:500 dilution (2.1 μg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). Tissue was counterstained with Hematoxylin. ImmunoHistoProbe one step HRP Polymer (ready to use) secondary antibody was used at 1:0 dilution. PBS instead of the primary antibody was used as the negative control.

All lanes : Anti-Caveolin-1 antibody [E249] - Caveolae Marker (ab32577) at 1/1000 dilution

Lane 1 : A431 cell lysate
Lane 2 : A549 cell lysate
Lane 3 : Wild-type HeLa cell lysate
Lane 4 : CAV1 knockout HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 20 kDa

Lanes 1 - 4: Merged signal (red and green). Green - ab32577 observed at 20 kDa. Red - loading control, ab8245 observed at 37 kDa.  

 ab32577 was shown to react with Caveolin-1 in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab255371 (knockout cell lysate ab263806) was used. Wild-type and Caveolin-1 knockout samples were subjected to SDS-PAGE. ab32577 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

 

Anti-Caveolin-1 antibody [E249] - Caveolae Marker (ab32577) + A431 cell lysate

Predicted band size: 20 kDa
Observed band size: 23 kDa why is the actual band size different from the predicted?
Additional bands at: 22 kDa. We are unsure as to the identity of these extra bands.

Immunocytochemistry/Immunofluorescence analysis of A431 cells labelling Caveolin-1 (green) with ab32577 at 1/200. Cells were fixed with 100% methanol. ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody. The cells were co-stained with ab195889 (red), an Alexa Fluor^® 488 conjugated mouse anti-tubulin antibody (1/200). Nuclei counterstained with DAPI (blue).

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Mouse testis tissue sections labeling Caveolin-1 with purified ab32577 at 1:500 dilution (2.1 μg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). Tissue was counterstained with Hematoxylin. ImmunoHistoProbe one step HRP Polymer (ready to use) secondary antibody was used at 1:0 dilution. PBS instead of the primary antibody was used as the negative control.

Immunohistochemical analysis of formalin-fixed, paraffin-embedded Human urinary bladder tissue, staining Caveolin-1 with ab32577 at 1/250 dilution.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

Immunohistochemical analysis of formalin-fixed, paraffin-embedded Human lung tissue, staining Caveolin-1 with ab32577 at 1/250 µg/ml.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

Immunohistochemical analysis of developing Human lung tissue, staining Caveolin-1 with ab32577 at 1/250 dilution.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.