Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: ARF6 knockout HAP1 cell lysate (20 µg)
Lane 3: HeLa cell lysate (20 µg)
Lane 4: HepG2 cell lysate (20 µg)
Lanes 1 - 4: Merged signal (red and green). Green - ab77581 observed at 18 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab77581 was shown to recognize ARF6 when ARF6 knockout samples were used, along with additional cross-reactive bands. Wild-type and ARF6 knockout samples were subjected to SDS-PAGE. ab77581 and ab8245 (loading control to GAPDH) were diluted 1 μg/mL and 1/10 000 respectively and incubated overnight at 4°C. 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 1/10 000 dilution for 1 h at room temperature before imaging.

IHC image of ARF6 staining in human liver carcinoma 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 (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab77581, 5µg/ml, for 15 mins 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.

All lanes : Anti-ARF6 antibody (ab77581) at 1 µg/ml

Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
Lane 2 : HEK293 (Human embryonic kidney cell line) Whole Cell Lysate
Lane 3 : HepG2 (Human hepatocellular liver carcinoma cell line) Whole Cell Lysate
Lane 4 : Human liver tissue lysate - total protein (ab29889)

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 20 kDa
Observed band size: 18 kDa why is the actual band size different from the predicted?



We hypothesize that the 18 kDa band represents the mature form of ARF6.

ICC/IF image of ab77581 stained HeLa cells. The cells were 4% PFA fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab77581, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM. This antibody also gave a positive result in 100% methanol fixed (5 min) HeLa cells at 1µg/ml.

All lanes : Anti-ARF6 antibody (ab77581) at 1 µg/ml

Lane 1 : Lysates prepared from Mouse liver tissue.
Lane 2 : Lysates prepared from Mouse lung tissue.
Lane 3 : Lysates prepared from Rat liver tissue.
Lane 4 : Lysates prepared from Rat lung tissue.

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : HRP-conjugated Pig polyclonal to rabbit IgG at 1/5000 dilution

Performed under reducing conditions.

Predicted band size: 20 kDa
Observed band size: 21 kDa why is the actual band size different from the predicted?


Exposure time: 2 minutes

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Immunohistochemical analysis of mouse Salivary Gland staining ARF6 with ab77581 at 1/50 dilution. An undiluted HRP conjugated Polyclonal anti rabbit was used as a secondary. Blocked with PB ab64226.

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