Recombinant Anti-DNA Ligase IV/LIG4 antibody [EPR16531] (ab193353)

Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: DNA Ligase IV/LIG4 knockout HAP1 cell lysate (20 µg)
Lane 3: HepG2 cell lysate (20 µg)
Lane 4: HeLa cell lysate (20 µg)
Lanes 1 - 4: Merged signal (red and green). Green - ab193353 observed at 103 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab193353 was shown to specifically react with DNA Ligase IV/LIG4 when DNA ligase IV knockout samples were used. Wild-type and DNA Ligase IV/LIG4 knockout samples were subjected to SDS-PAGE. ab193353 and ab8245 (loading control to GAPDH) were diluted 1/1000 and 1/2000 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.

ab193353 staining DNA Ligase IV/LIG4 in wild-type HAP1 cells (top panel) and LIG4 knockout HAP1 cells (bottom panel). The cells were fixed with 4% formaldehyde (10min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab193353 at 5μg/ml dilution and ab195889 at 1/250 dilution (shown in pseudo colour red) overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to Rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green). Nuclear DNA was labelled in blue with DAPI.

Immunohistochemical analysis of paraffin embedded Human thymus tissue sections labeling DNA Ligase IV/LIG4 using ab193353 at a 1/50 dilution. A ready to use HRP Polymer for Rabbit IgG was used as the secondary. Hematoxylin counterstain.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunofluorescent analysis of 4% paraformaldehyde fixed SH-SY5Y cells labeling DNA Ligase IV/LIG4 using ab193353 at a 1/50 dilution. A Goat anti rabbit IgG (Alexa Fluor®488) (ab150077) was used as the secondary at a 1/400 dilution. Counterstain DAPI. Cells were permeabilized using 0.1% Triton X-100.