Anti-Caveolin-1 antibody [EPR15554] - BSA and Azide free

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-Caveolin-1 antibody [EPR15554] - BSA and Azide free (AB240332)

This data was developed using the same antibody clone in a different buffer formulation (ab192869).

ab192869 staining Caveolin-1 in wild-type HeLa cells (top panel) and CAV1 knockout HeLa cells (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).

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-Caveolin-1 antibody [EPR15554] - BSA and Azide free (AB240332)

Intracellular Intracellular Flow Cytometry overlay histogram showing wild-type HeLa (green line) and CAV1 knockout HeLa cells (ab255371) stained with ab240332 (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 (ab240332) (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.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Caveolin-1 antibody [EPR15554] - BSA and Azide free (AB240332)

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).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab192869).

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Caveolin-1 antibody [EPR15554] - BSA and Azide free (AB240332)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab192869).

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

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Caveolin-1 antibody [EPR15554] - BSA and Azide free (AB240332)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab192869).

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

• IP

Supplier Data

Immunoprecipitation - Anti-Caveolin-1 antibody [EPR15554] - BSA and Azide free (AB240332)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab192869).

All lanes:

Immunoprecipitation - Anti-Caveolin-1 antibody [EPR15554] - N-terminal - Caveolae Marker (<a href='/en-us/products/primary-antibodies/caveolin-1-antibody-epr15554-n-terminal-caveolae-marker-ab192869'>ab192869</a>)

Predicted band size: 20 kDa

false

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Caveolin-1 antibody [EPR15554] - BSA and Azide free (AB240332)

Immunohistochemical analysis of paraffin-embedded Mouse 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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab192869).

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

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-Caveolin-1 antibody [EPR15554] - BSA and Azide free (AB240332)

Intracellular 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).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab192869).

• WB

Lab

Western blot - Anti-Caveolin-1 antibody [EPR15554] - BSA and Azide free (AB240332)

This data was developed using the same antibody clone in a different buffer formulation (ab192869).

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 A431 wild-type 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 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.

All lanes:

Western blot - Anti-Caveolin-1 antibody [EPR15554] - N-terminal - Caveolae Marker (<a href='/en-us/products/primary-antibodies/caveolin-1-antibody-epr15554-n-terminal-caveolae-marker-ab192869'>ab192869</a>) at 1/1000 dilution

Lane 1:

Wild-type A-431 (Human epidermoid carcinoma cell line) whole cell lysate at 20 µg

Lane 2:

CAV1 knockout A-431 (Human epidermoid carcinoma cell line) whole cell lysate at 20 µg

Lane 2:

Western blot - Human CAV1 (Caveolin-1) knockout A-431 cell line (<a href='/en-us/products/cell-lines/human-cav1-caveolin-1-knockout-a-431-cell-line-ab269583'>ab269583</a>)

Lane 3:

A549 (Human lung carcinoma cell line) whole cell lysate at 20 µg

Lane 4:

HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg

Predicted band size: 20 kDa

Observed band size: 21-24 kDa

false

• WB

Lab

Western blot - Anti-Caveolin-1 antibody [EPR15554] - BSA and Azide free (AB240332)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab192869).

Western blot : Anti-Caveolin-1 antibody [EPR15554] - N-terminal - Caveolae Marker ab192869 staining at 1/1000 dilution, shown in green; Mouse anti alpha Tubulin ab7291 loading control staining at 1/20000 dilution, shown in magenta. A band was observed at 20 kDa in Wild-type HeLa cell lysates with no signal observed at this size in CAV1 knockout HeLa cell line. To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 5pc 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 800CW and Goat anti-Mouse 680RD at 1/20,000 dilution.

All lanes:

Western blot - Anti-Caveolin-1 antibody [EPR15554] - N-terminal - Caveolae Marker (<a href='/en-us/products/primary-antibodies/caveolin-1-antibody-epr15554-n-terminal-caveolae-marker-ab192869'>ab192869</a>) at 1/1000 dilution

Lane 1:

Wild-type HeLa whole cell lysate at 20 µg

Lane 2:

CAV1 knockout HeLa whole cell lysate at 20 µg

Lane 3:

Wild-type U-87 MG ab278079 whole cell lysate at 20 µg

Lane 4:

Western blot - Human CAV1 knockout U-87 MG cell line (ab306811) at 20 µg

Lane 5:

A431 whole cell lysate at 20 µg

Lane 6:

Ramos whole cell lysate at 20 µg

Lane 7:

Raji whole cell lysate at 20 µg

Lane 8:

HepG2 whole cell lysate at 20 µg

Secondary

All lanes:

Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20000 dilution

Predicted band size: 20 kDa

Observed band size: 20 kDa

false

• WB

Lab

Western blot - Anti-Caveolin-1 antibody [EPR15554] - BSA and Azide free (AB240332)

This data was developed using the same antibody clone in a different buffer formulation (ab192869)

ab240332 was shown to react with CAV1 in wild-type U-87 MG cells in Western blot with loss of signal observed in CAV1 knockout cell line ab306811. Wild-type U-87 MG and CAV1 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 5% milk in TBST for 1 hr before incubation with ab192869 overnight at 4 °C at a 1/10000 dilution. Blots were incubated with secondary antibodies at 0.2 µg/mL before imaging.

This data was provided by YCharOS Inc., an open science company with the mission of characterizing commercially available antibody reagents for all human proteins. Abcam and YCharOS are working together to help address the reproducibility crisis by enabling the life science community to better evaluate commercially available antibodies.

All lanes:

Western blot - Anti-Caveolin-1 antibody [EPR15554] - N-terminal - Caveolae Marker (<a href='/en-us/products/primary-antibodies/caveolin-1-antibody-epr15554-n-terminal-caveolae-marker-ab192869'>ab192869</a>) at 1/10000 dilution

Lane 1:

Wild-type U-87 MG lysate at 20 µg

Lane 2:

CAV1 knock-out U-87 MG lysate at 20 µg

Lane 2:

Western blot - Human CAV1 knockout U-87 MG cell line (ab306811)

Observed band size: 22 kDa

false

• WB

Lab

Western blot - Anti-Caveolin-1 antibody [EPR15554] - BSA and Azide free (AB240332)

This data was developed using the same antibody clone in a different buffer formulation (ab192869).

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:

Western blot - Anti-Caveolin-1 antibody [EPR15554] - BSA and Azide free (ab240332) at 1/10000 dilution

Lane 1:

A431 cell lysate at 20 µg

Lane 2:

A549 cell lysate at 20 µg

Lane 3:

Wild-type HeLa cell lysate at 20 µg

Lane 4:

Caveolin-1 knockout HeLa cell lysate at 20 µg

Predicted band size: 20 kDa

Observed band size: 20 kDa

false