ARL3 Antibody / ADP-ribosylation factor-like protein 3 (FY12429)

Immunofluorescent staining of using anti-ARL3 antibody (green) and anti-Beta Tubulin antibody (red). ARL3 was detected in an immunocytochemical section of human HeLa cells. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent for 15 mins. The cells were blocked with 10% goat serum. And then incubated with 5 ug/ml rabbit anti-ARL3 antibody and mouse anti-Beta Tubulin antibody overnight at 4oC. DyLight 488 Conjugated Goat Anti-Rabbit IgG and Cy3 Conjugated Goat Anti-Mouse IgG were used as secondary antibody at 1:500 dilution and incubated for 30 minutes at 37oC. Visualize using a fluorescence microscope and filter sets appropriate for the label used.

Western blot analysis of using anti-ARL3 antibody. Lane 1: human Hela whole cell lysates, Lane 2: human whole cell lysates, Lane 3: rat testis tissue lysates, Lane 4: mouse testis tissue lysates. After electrophoresis, proteins were transferred to a nitrocellulose membrane at 150 mA for 50-90 minutes. Blocked the membrane with 5% non-fat milk/TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti- antibody at 0.5 ug/ml overnight at 4oC, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-ARL3 rabbit IgG-HRP secondary antibody at a dilution of 1:5000 for 1.5 hour at RT. The signal was developed using enhanced chemiluminescent. ARL3 (~20 kDa predicted) was detected as a doublet at ~20-23 kDa, consistent with the coexistence of non-myristoylated and myristoylated species described in previous studies.

Flow Cytometry analysis of human SH-SY5Y cells using anti-ARL3 antibody. Overlay histogram showing SH-SY5Y cells stained with (Blue line). To facilitate intracellular staining, cells were fixed with 4% paraformaldehyde and permeabilized with permeabilization buffer. The cells were blocked with 10% normal goat serum. And then incubated with rabbit anti-ARL3 antibody (1 ug/million cells) for 30 min at 20oC. DyLight 488 conjugated goat anti-rabbit IgG (5-10 ug/million cells) was used as secondary antibody for 30 minutes at 20oC. Isotype control antibody (Green line) was rabbit IgG (1 ug/million cells) used under the same conditions. Unlabelled sample without incubation with primary antibody and secondary antibody (Red line) was used as a blank control.