Anti-HEF1/NEDD-9 抗体 [2G9]

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-HEF1/NEDD-9 antibody [2G9] (AB18056)

Immunofluorescence staining of MDA-MB231 cells with the 2G9 antibody at a 1/200 dilution. Cells were fixed in 3.8% PFA for 10 minutes, and staining was performed for 1 hour at room temperature.

• ICC

Lab

Immunocytochemistry - Anti-HEF1/NEDD-9 antibody [2G9] (AB18056)

ICC/IF image of ab18056 stained MCF7 (human breast adenocarcinoma cell line) cells. The cells were fixed in 4% formaldehyde for 10 minutes and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1 hour to permeabilize the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab18056, 1 μg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor^® 488 goat anti-mouse IgG (H+L) used at a 1/1000 dilution for 1 hour. Alexa Fluor^® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1 hour. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43 μM.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-HEF1/NEDD-9 antibody [2G9] (AB18056)

Immunofluorescence analysis of paraformaldehyde fixed MCF7 cells, permeabilized with 0.15% Triton.

Primary incubation 1hr (1 : 100 dilution) followed by Alexa Fluor® 488 secondary antibody (1 : 1000 dilution), showing cytoplasmic staining. The nuclear stain is DAPI (blue).

Negative control : Mouse IgG1 negative control followed by Alexa Fluor® 488 secondary antibody.

• Flow Cyt

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Flow Cytometry - Anti-HEF1/NEDD-9 antibody [2G9] (AB18056)

Overlay histogram showing A549 (human lung carcinoma cell line) cells stained with ab18056 (red line). The cells were fixed with 80% methanol (5 minutes) and then permeabilized with 0.1% PBS-Tween for 20 minutes. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab18056, 1 μg/ 1 x 10⁶ cells) for 30 minutes at 22°C. The secondary antibody used was DyLight^® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 minutes at 22°C. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2 μg/ 1 x 10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in A549 cells fixed with 4% paraformaldehyde (10 minutes)/permeabilized in 0.1% PBS-Tween used under the same conditions.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HEF1/NEDD-9 antibody [2G9] (AB18056)

IHC image of ab18056 staining in human kidney formalin fixed paraffin embedded tissue section, performed on a Leica Bond^TM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6, epitope retrieval solution 1) for 20 minutes. The section was then incubated with ab18056, 10 μg/ml, for 15 minutes at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

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.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-HEF1/NEDD-9 antibody [2G9] (AB18056)

Immunofluorescence analysis of paraformaldehyde fixed MCF7 cells, permeabilized with 0.15% Triton.

Primary incubation 1hr (1 : 100 dilution) followed by Alexa Fluor® 488 secondary antibody (1 : 1000 dilution), showing cytoplasmic staining. The nuclear stain is DAPI (blue).

Negative control : Mouse IgG1 negative control followed by Alexa Fluor® 488 secondary antibody.

• WB

Lab

Western blot - Anti-HEF1/NEDD-9 antibody [2G9] (AB18056)

Western blot : Anti-HEF1/NEDD-9 antibody [2G9] ab18056 staining at 0.1 µg/mL, shown in green; Rabbit anti alpha Tubulin (ab52866) loading control staining at 1/20,000 dilution, shown in magenta. A band was observed at 100 kDa in Wild-type A549 cell lysates with no signal observed at this size in NEDD9 knockout A549 cell line.

To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3pc 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-Mouse 800CW & Goat anti-Rabbit 680RD at 1/20,000 dilution.

All lanes:

Western blot - Anti-HEF1/NEDD-9 antibody [2G9] (ab18056) at 0.1 µg/mL

Lane 1:

Wild-type A549 at 20 µg

Lane 2:

NEDD9 knockout A549 at 20 µg

Lane 3:

HepG2 at 20 µg

Lane 4:

PANC-1 at 20 µg

Secondary

All lanes:

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

Predicted band size: 93 kDa

Observed band size: 100 kDa

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• WB

Lab

Western blot - Anti-HEF1/NEDD-9 antibody [2G9] (AB18056)

(0.1µg/ml) staining in MCF7 cells lysate (35µg protein in RIPA buffer). Primary incubation was 1 hour. Detected by chemiluminescence.

All lanes:

Western blot - Anti-HEF1/NEDD-9 antibody [2G9] (ab18056) at 0.1 µg/mL

Predicted band size: 92 kDa

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