Anti-ARF6 antibody (ab77581)

Western blot - ARF6 antibody (ab77581)

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 : Liver (Human) Tissue Lysate - adult normal tissue (ab29889)

Lysates/proteins at 10 µg per lane.

Secondary
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.

Immunocytochemistry/ Immunofluorescence - ARF6 antibody (ab77581)

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 +4ºC. 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.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - ARF6 antibody (ab77581)

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.

Western blot - ARF6 antibody (ab77581)

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
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

This image is a courtesy of Antibody Solutions Ltd