Anti-Annexin V/ANXA5 antibody [EPR3980]

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-Annexin V/ANXA5 antibody [EPR3980] (AB108194)

Flow Cytometry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labelling Annexin V/ANXA5 with purified ab108194 at 1/30 dilution (10 µg/ml) (red). Cells were fixed with 80% Methanol and permeabilised with 0.1% Tween-20. A Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150081) secondary antibody was used at 1/2000. Isotype control - Rabbit monoclonal IgG (black). Unlabelled control - Cell without incubation with primary antibody and secondary antibody (blue).

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Annexin V/ANXA5 antibody [EPR3980] (AB108194)

Immunocytochemistry analysis of JAR (Human placenta choriocarcinoma epithelial cell) cells labeling Annexin V/ANXA5 with purified ab108194 at 1 : 50 dilution (2.2 µg/ml). Cells were fixed in 100% Methanol and permeabilized with 0.1% tritonX-100. Cells were counterstained with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) at 1/200 dilution (2.5 µg/ml) (ab195889) (red). Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) was used as the secondary antibody at 1 : 1000 (2 µg/ml) dilution. DAPI (blue) was used as a nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

• WB

Supplier Data

Western blot - Anti-Annexin V/ANXA5 antibody [EPR3980] (AB108194)

All lanes:

Western blot - Anti-Annexin V/ANXA5 antibody [EPR3980] (ab108194) at 1/10000 dilution

Lane 1:

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

Lane 2:

Jurkat (Human T cell leukemia T lymphocyte) whole cell lysate at 20 µg

Lane 3:

Mouse brain at 20 µg

Lane 4:

Mouse heart at 20 µg

Lane 5:

Rat brain at 20 µg

Lane 6:

Rat heart at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 35 kDa

Observed band size: 36 kDa

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• WB

Lab

Western blot - Anti-Annexin V/ANXA5 antibody [EPR3980] (AB108194)

Lane 1 : Wild-type HAP1 whole cell lysate (20 μg)
Lane 2 : Annexin V knockout HAP1 whole cell lysate (20 μg)
Lane 3 : HeLa whole cell lysate (20 μg)

Lanes 1 - 3 : Merged signal (red and green). Green - ab108194 observed at 35 kDa. Red - loading control, ab18058, observed at 130 kDa.

ab108194 was shown to recognize Annexin V in wild-type cells as signal was lost at the expected MW in Annexin V knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and Annexin V knockout samples were subjected to SDS-PAGE. ab108194 and ab18058 (Mouse anti-Vinculin loading control) were incubated overnight at 4°C at 1/10000 dilution and 1/20000 dilution respectively. Blots were developed 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 1/20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-Annexin V/ANXA5 antibody [EPR3980] (ab108194)

Predicted band size: 35 kDa

false

• WB

Lab

Western blot - Anti-Annexin V/ANXA5 antibody [EPR3980] (AB108194)

Western blot : Anti-ANXA5 antibody [EPR3980] (ab108194) staining at 1/10000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab108194 was shown to bind specifically to ANXA5. A band was observed at 36 kDa in wild-type A549 cell lysates with no signal observed at this size in ANXA5 knockout cell line. To generate this image, wild-type and ANXA5 knockout A549 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % 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 IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-Annexin V/ANXA5 antibody [EPR3980] (ab108194) at 1/10000 dilution

Lane 1:

Wild-type A549 cell lysate at 20 µg

Lane 2:

Western blot - Human ANXA5 knockout A549 cell line (<a href='/en-us/products/cell-lines/human-anxa5-knockout-a549-cell-line-ab300840'>ab300840</a>)

Lane 2:

ANXA5 knockout A549 cell lysate at 20 µg

Secondary

Lanes 1 - 2:

Goat anti-Rabbit IgG H&L 800CW at 1/20000 dilution

Lanes 1 - 2:

Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Observed band size: 36 kDa

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