All lanes : Anti-Caveolin-1 antibody [7C8] (ab17052) at 1 µg/ml

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

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

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 - ab17052 observed at 21-24 kDa. Red - loading control, ab52866 (Rabbit anti-alpha Tubulin antibody [EP1332Y]) observed at 55kDa.

ab17052 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 non-mammalian (TBS-based) blocking solution before incubation with ab17052 and ab52866 (Rabbit anti-alpha Tubulin antibody [EP1332Y]) overnight at 4°C at 1 µg/ml and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Mouse IgG H&L (IRDye^® 800CW) preabsorbed (ab216772) and Goat anti-Rabbit IgG H&L (IRDye^® 680RD) preabsorbed (ab216777) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

ab17052 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 ab17052 at 1/500 dilution and ab6046 (Rabbit polyclonal to beta 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 mouse IgG (Alexa Fluor^® 488) (ab150117) at 2 μg/ml (shown in green) and a goat secondary antibody to rabbit IgG (Alexa Fluor^® 594) (ab150080) 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).

 ab17052 staining Caveolin-1 in human Hacat keratinocyte cells by Immunocytochemistry/ Immunofluorescence. The cells were formaldehyde fixed, permeabilised in 0.1% Triton X-100 and then blocked using 1% serum for 1 hour at 25°C. Samples were then incubated with primary antibody at 1/75 for 24 hours at 4°C. The secondary antibody used was conjugated to Alexa Fluor® 488 (green) used at a 1/500 dilution.

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Anti-Caveolin-1 antibody [7C8] (ab17052) at 1/500 dilution + HUVEC whole cell lysate at 20 µg

Developed using the ECL technique.

Performed under reducing conditions.

Exposure time: 10 seconds

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Immunocytochemical analysis of Mouse Bend.3 cells, labeling Caveolin-1 with ab17052. Cells were paraformaldehyde fixed, permeabilized with PBS + 0.1% Triton (PBT), and blocked with 10% serum for 1 hour at 22°C. Staining with ab17052 (diluted 1/200) was for 16 hours at 4°C.

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ab17052 at a 1/500 dilution staining hamster CHO cells by ICC/IF. The cells were paraformaldehyde fixed and blocked with 10% serum prior to incubation with the antibody. Bound antibody was detected using a FITC conjugated goat anti-mouse antibody.

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