LIG4 Antibody / DNA Ligase 4 (FY13129)

Immunofluorescent staining of DNA Ligase IV/LIG4 using anti-LIG4 antibody (green) and anti-Tubulin Alpha antibody (red). DNA Ligase IV/LIG4 was detected in immunocytochemical section of 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-DNA Ligase IV/LIG4 antibody and mouse anti-Tubulin Alpha 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 LIG4 using anti-LIG4 antibody. Electrophoresis was performed on a 8% SDS-PAGE gel at 80V (Stacking gel) / 120V (Resolving gel) for 2 hours. Lane 1: human Ramos whole cell lysates, Lane 2: human HepG2 whole cell lysates, Lane 3: human jurkat whole cell lysates, Lane 4: human Hela whole cell lysates, Lane 5: rat testis tissue lysates, Lane 6: 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-LIG4 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 an ECL Plus Western Blotting Substrate. LIG4 antibody detects a primary band at ~110 kDa across the indicated human and rodent lysates, consistent with full-length DNA ligase IV. Although the theoretical mass is ~104 kDa, slower migration is typical due to phosphorylation and the acidic C-terminal domain. Additional weaker bands just below 100 kDa likely represent partially dephosphorylated or proteolytically processed forms of LIG4.

Flow Cytometry analysis of Jurkat cells using anti-LIG4 antibody. Overlay histogram showing Jurkat 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-LIG4 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.