All lanes : Anti-Caveolin-1 antibody [EPR15554] - N-terminal (ab192869) 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: 17, 20 kDa
Observed band size: 21-24 kDa why is the actual band size different from the predicted?

Lanes 1 - 4: Merged signal (red and green). Green - ab192869 observed at 21-24 kDa. Red - loading control, ab7291 (Mouse anti-Alpha Tubulin [DM1A] observed at 55kDa.

ab192869 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. Wild-type and CAV1 knockout A431 cell lysates were subjected to SDS-PAGE. Membranes were blocked in 5% milk in TBS-T (0.1% Tween^®) before incubation with ab192869 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.

ab192869 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 ab192869 at 1/500 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).

Immunohistochemical analysis of paraffin-embedded Human squamous cell carcinoma of cervix tissue labeling Caveolin-1 using ab192869 at 1/4000 dilution (0.15 μg/ml). A prediluted HRP Polymer for Rabbit IgG was used as the secondary. Counterstain: Hematoxylin. Negative control also shown.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Flow cytometry overlay histogram showing wild-type HeLa (green line) and CAV1 knockout HeLa cells (ab255371) stained with ab192869 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction followed by the antibody (ab192869) (1x10⁶ in 100μl at 0.04 μg/ml) for 30 min at 22°C.

The secondary antibody Goat anti-rabbit IgG H&L (Alexa Fluor^® 488, pre-adsorbed) (ab150081) was used at 1/2000 for 30 min at 22°C.

Isotype control antibody was Rabbit IgG (monoclonal) (ab172730) used at the same concentration and conditions as the primary antibody (wild-type HeLa - black line CAV1 knockout HeLa - grey line). Unlabelled sample was also used as a control (this line is not shown for the purpose of simplicity).

Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter.

All lanes : Anti-Caveolin-1 antibody [EPR15554] - N-terminal (ab192869) at 1/10000 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: 17, 20 kDa

Lanes 1 - 4: Merged signal (red and green). Green - ab192869 observed at 20 kDa. Red - loading control, ab8245 observed at 37 kDa.  

 ab192869 was shown to react with Caveolin-1 in wild-type HeLa. 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. ab192869 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 10000 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.

 

All lanes : Anti-Caveolin-1 antibody [EPR15554] - N-terminal (ab192869) at 1/10000 dilution

Lane 1 : PC-3 cell lysate
Lane 2 : A549 cell lysate
Lane 3 : A431 cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Predicted band size: 17, 20 kDa

Immunoprecipitation analysis of A431 cell lysate labeling Caveolin-1 using ab192869 at 1/30 dilution (Lane 1). PBS negative control (Lane 2). Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/1500 was used as the secondary antibody.

Immunohistochemical analysis of paraffin-embeddedMouse lung tissue labeling Caveolin-1 using ab192869 at 1/4000 dilution (0.15 μg/ml). A prediluted HRP Polymer for Rabbit IgG was used as the secondary. Counterstain: Hematoxylin. Negative control also shown.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded Human liver tissue labeling Caveolin-1 using ab192869 at 1/4000 dilution (0.15 μg/ml). A prediluted HRP Polymer for Rabbit IgG was used as the secondary. Counterstain: Hematoxylin. Negative control also shown.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunocytochemistry/Immunofluorescence analysis of A-673 cells labelling Caveolin-1 with ab192869 at a dilution of 1/100. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. 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, Alexa Fluor^® 594-conjugated anti-Tubulin [DM1A] at a dilution of 1/200. Nuclei counterstained with DAPI (blue).

Flow cytometry analysis of NIH3T3 cells labeling Caveolin-1 using ab192869 at a 1/120 dilution (Red). A Goat anti rabbit IgG (FITC) at 1/150 dilution was used as secondary antibody. Cells were fixed with 2% paraformaldehyde. Cells without incubation with primary antibody and secondary antibody Blue. Rabbit monoclonal IgG was used as isotype control (Black).