All lanes : Anti-Axl antibody [EPR19880] (ab219651) at 1/10000 dilution

Lane 1 : Wild-type HeLa cell lysate at 40 µg
Lanes 2 & 4 : AXL knockout HeLa cell lysate at 20 µg
Lane 3 : Wild-type HeLa cell lysate at 20 µg

Performed under reducing conditions.

Predicted band size: 98 kDa
Observed band size: 135 kDa why is the actual band size different from the predicted?

Lanes 1 - 4: Merged signal (red and green). Green - ab219651 observed at 135 kDa. Red - loading control, ab7291 (Mouse anti-Alpha Tubulin [DM1A]) observed at 55kDa.

ab219651 was shown to react with Axl in western blot. Membranes were blocked in 3% milk in TBS-T (0.1% Tween^®) before incubation with ab219651 and ab7291 (Mouse anti-Alpha Tubulin [DM1A]) overnight at 4°C at a 1 in 10000 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.

Immunohistochemical analysis of paraffin-embedded human breast and breast carcinoma tissue labeling Axl with ab219651 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Cytoplasmic staining on cancer cells of the human breast carcinoma, and the pericarcinous tissue was nearly negative (magnification x150) (PMID: 25337673).

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

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

Immunohistochemical analysis of paraffin-embedded human lung tissue labeling Axl with ab219651 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Sporadic cytoplasmic staining on human lung.

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

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

Immunohistochemical analysis of paraffin-embedded human lung carcinoma tissue labeling Axl with ab219651 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Cytoplasmic staining on cancer cells of human lung carcinoma (PMID: 25337673).

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

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

Immunohistochemical analysis of paraffin-embedded human adipose tissue labeling Axl with ab219651 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Negative control: No staining on human adipose tissues (PMID: 26573603).

Counter stained with Hematoxylin.

 

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

All lanes : Anti-Axl antibody [EPR19880] (ab219651) at 1/10000 dilution

Lane 1 : 293T (Human epithelial cell line from embryonic kidney) transfected with an empty expression vector, whole cell lysate
Lane 2 : 293T (Human epithelial cell line from embryonic kidney) transfected with a His-tagged human Axl construct whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase at 1/100000 dilution

Predicted band size: 98 kDa
Observed band size: 138 kDa why is the actual band size different from the predicted?


Exposure time: 5 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

Detection of endogenous expression of Axl in WB will need optimization. Under our experimental conditions we could not detect endogenous Axl in human cell and tissue lysates.