Anti-Axl antibody [EPR23892-15]

• Flow Cyt

Supplier Data

Flow Cytometry - Anti-Axl antibody [EPR23892-15] (AB259831)

Flow cytometric analysis of Parental HeLa (human cervix adenocarcinoma epithelial cell (Right panel)/ AXL knockout HeLa cells (Left panel) cells labelling Axl with ab259831 at 1/500 dilution (0.1μg) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

Positive staining on HeLa cells (ab255928), while no staining on AXL knockout HeLa cells (ab261810).

Gated on viable cells.

• Flow Cyt

Supplier Data

Flow Cytometry - Anti-Axl antibody [EPR23892-15] (AB259831)

Flow cytometric analysis of Jurkat (Human T cell leukemia T lymphocyte, Left panel)/ NCI-H1299 (human lung carcinoma epithelial cell, Right panel) cells labelling Axl with ab259831 at 1/500 dilution (0.1μg) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

Negative control : Jurkat (PMID : 28423548).

Gated on viable cells.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Axl antibody [EPR23892-15] (AB259831)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized Axl KO HeLa (ab261810) cells labelling Axl with ab259831 at 1/100 (4.9 μg/ml) dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) antibody at 1/1000 dilution (Green). Confocal image showing strong membranous and weak cytoplasmic staining in parental HeLa cells (ab255928), while no staining in AXL knockout HeLa cells (ab261810). ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 dilution (Red). The nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 dilution.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Axl antibody [EPR23892-15] (AB259831)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Trito nX-100 permeabilized NCI-H1299 cells labelling Axl with ab259831 at 1/500 (0.98 μg/ml) dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) antibody at 1/1000 dilution (Green). Confocal image showing strong membranous and weak cytoplasmic staining in NCI-H1299 cells (PMID : 27443522). ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 dilution (Red). The nuclear counterstain was DAPI (Blue).

Negative control : Jurkat (PMID : 28423548).

Secondary antibody only control : Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 dilution.

• WB

Lab

Western blot - Anti-Axl antibody [EPR23892-15] (AB259831)

Blocking and diluting buffer and concentration : 5% NFDM/TBST

Negative control : Jurkat (PMID : 28423548).

Exposure time : 37 seconds

All lanes:

Western blot - Anti-Axl antibody [EPR23892-15] (ab259831) at 1/1000 dilution

Lane 1:

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

Lane 2:

DU145 (human prostate carcinoma epithelial cell), whole cell lysate at 20 µg

Lane 3:

NCI-H1299 (human lung carcinoma epithelial cell), whole cell lysate at 20 µg

Lane 4:

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

Secondary

All lanes:

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

Predicted band size: 98 kDa

Observed band size: 140 kDa,80 kDa

false

• WB

Lab

Western blot - Anti-Axl antibody [EPR23892-15] (AB259831)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

ab219651 exhibits low sensitivity in western blot. It is recommended to use ab259831 or ab215205 as alternatives or to optimize experimental conditions by increasing the sample loading amount, using a lower antibody dilution ratio, and employing femtogram-level sensitivity substrates.

Lanes 1 - 3:

Western blot - Anti-Axl antibody [EPR19880] (<a href='/en-us/products/primary-antibodies/axl-antibody-epr19880-ab219651'>ab219651</a>) at 1/1000 dilution

Lanes 5 - 7:

Western blot - Anti-Axl antibody [EPR23892-15] (ab259831) at 1/1000 dilution

Lanes 9 - 11:

Western blot - Anti-Axl antibody [EPR21107] (<a href='/en-us/products/primary-antibodies/axl-antibody-epr21107-ab215205'>ab215205</a>) at 1/1000 dilution

Lanes 1, 5 and 9:

HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate 20 µg

Lanes 2, 6 and 9:

HUVEC (human umbilical vein endothelial cell line) whole cell lysate 20 µg

Lanes 3, 7 and 11:

A549 (Human lung carcinoma epithelial cell) whole cell lysate 20 µg

Lanes 4, 8 and 12:

Jurkat (human T cell leukemia cell line from peripheral blood) whole cell lysate 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: 98 kDa

Observed band size: 80 kDa,140 kDa

false

Exposure time: 180s

• WB

Lab

Western blot - Anti-Axl antibody [EPR23892-15] (AB259831)

Blocking and diluting buffer and concentration : Intercept^® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS

Lanes 1-4 : Merged signal (red and green). Green - ab259831 observed at 140, 80 kDa. Red - loading control ab8245 observed at 36 kDa.

ab259831 Anti-Axl antibody [EPR23892-15] was shown to react with Axl in Hela cells in Western blot. Loss of signal was observed when knockout cell line ab261810 (knockout cell lysate ab257151) was used. Wild-type and Axl knockout samples were subjected to SDS-PAGE.

ab259831 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated at 4°C overnight at 1 in 1000 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 10000 dilution for 1 hour at room temperature before imaging.

Negative control : Jurkat (PMID : 28423548).

Observed band size are 140,80 kDa. The 80 kDa band is the soluble Axl (PMID : 7822279)

All lanes:

Western blot - Anti-Axl antibody [EPR23892-15] (ab259831) at 1/1000 dilution

Lane 1:

Wild-type HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 40 µg

Lane 2:

Axl knockout HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 40 µg

Lane 3:

NCI-H1299 (human lung carcinoma epithelial cell) whole cell lysate at 40 µg

Lane 4:

Jurkat (human T cell leukemia T lymphocyte), whole cell lysate at 40 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG H&L (IRDye® 800CW) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) and Goat Anti-Mouse IgG H&L (IRDye® 680RD) (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-h-l-irdye-680rd-preadsorbed-ab216776'>ab216776</a>) at 1/10000 dilution

Predicted band size: 98 kDa

Observed band size: 140 kDa,80 kDa

false

• WB

Lab

Western blot - Anti-Axl antibody [EPR23892-15] (AB259831)

Blocking and diluting buffer and concentration : Intercept^® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS

Lanes 1-4 : Merged signal (red and green). Green - ab259831 observed at 140, 80 kDa. Red - loading control ab8245 observed at 36 kDa.

ab259831 Anti-Axl antibody [EPR23892-15] was shown to react with Axl in Hela cells in Western blot. Loss of signal was observed when knockout cell line ab261810 (knockout cell lysate ab257151) was used. Wild-type and Axl knockout samples were subjected to SDS-PAGE.

ab259831 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated at 4°C overnight at 1 in 1000 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 10000 dilution for 1 hour at room temperature before imaging.

Negative control : Jurkat (PMID : 28423548).

Observed band size are 140,80 kDa. The 80 kDa band is the soluble Axl (PMID : 7822279)

All lanes:

Western blot - Anti-Axl antibody [EPR23892-15] (ab259831) at 1/1000 dilution

Lane 1:

Wild-type HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 40 µg

Lane 2:

Axl knockout HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 40 µg

Lane 2:

Western blot - Human AXL knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-axl-knockout-hela-cell-line-ab261810'>ab261810</a>)

Lane 3:

NCI-H1299 (human lung carcinoma epithelial cell) whole cell lysate at 40 µg

Lane 4:

Jurkat (human T cell leukemia T lymphocyte), whole cell lysate at 40 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG H&L (IRDye® 800CW) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) and Goat Anti-Mouse IgG H&L (IRDye® 680RD) (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-h-l-irdye-680rd-preadsorbed-ab216776'>ab216776</a>) at 1/10000 dilution

Predicted band size: 98 kDa

Observed band size: 140 kDa,80 kDa

false

• WB

Lab

Western blot - Anti-Axl antibody [EPR23892-15] (AB259831)

False colour image of Western blot : Anti-Axl antibody [EPR23892-15] staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab259831 was shown to bind specifically to Axl. A band was observed at 135 kDa in wild-type A549 cell lysates with no signal observed at this size in AXL knockout cell line ab273744 (knockout cell lysate ab273780). To generate this image, wild-type and AXL 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. Observed band size are 140,80 kDa. The 80 kDa band is the soluble Axl (PMID : 7822279)

All lanes:

Western blot - Anti-Axl antibody [EPR23892-15] (ab259831) at 1/1000 dilution

Lane 1:

Wild-type A549 cell lysate at 20 µg

Lane 2:

AXL knockout A549 cell lysate at 20 µg

Lane 3:

A549 cell lysate at 20 µg

Lane 4:

Jurkat cell lysate at 20 µg

Predicted band size: 98 kDa

Observed band size: 135 kDa

false