SIRT6 Antibody / Sirtuin 6 (FY13069)

Immunofluorescent staining of SIRT6 using anti-SIRT6 antibody (red) and anti-Beta Tubulin antibody (green). SIRT6 was detected in immunocytochemical section of U2OS cell. 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-SIRT6 antibody and mouse anti-Beta Tubulin antibody overnight at 4oC. Cy3 Conjugated Goat Anti-Rabbit IgG and DyLight 488 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 SIRT6 using anti-SIRT6 antibody. Lane 1: human Hela whole cell lysates, Lane 2: human Jurkat whole cell lysates, Lane 3: human 293T whole cell lysates, Lane 4: human K562 whole cell 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-SIRT6 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-rabbit IgG-HRP secondary antibody at a dilution of 1:5000 for 1.5 hour at RT. The signal was developed using enhanced chemiluminescent. A prominent band is detected at approximately 42 kDa, consistent with the reported migration of SIRT6, which typically runs above its predicted 36-39 kDa size. The apparent doublet pattern reflects the coexistence of two SIRT6 isoforms and/or post-translationally modified species such as phosphorylated or acetylated forms. Minor bands slightly above or below this range are occasionally observed and correspond to differentially modified or partially processed variants of SIRT6 described in the literature.

Flow Cytometry analysis of Hela cells using anti-SIRT6 antibody. Overlay histogram showing Hela 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-SIRT6 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 (Red line) was also used as a control.