Western blot analysis of extracts from various cell lines using DNA Ligase IV (D5N5N) Rabbit mAb.
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.
For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.
NOTE: Please refer to primary antibody product webpage for recommended antibody dilution.
From sample preparation to detection, the reagents you need for your Western Blot are now in one convenient kit: #12957 Western Blotting Application Solutions Kit
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).
NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.
* Avoid repeated exposure to skin.
posted June 2005
revised June 2020
Protocol Id: 10
DNA Ligase IV (D5N5N) Rabbit mAb recognizes endogenous levels of total DNA ligase IV protein.
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Leu771 of human DNA ligase IV protein.
DNA double-strand breaks (DSBs) are potentially hazardous lesions that can be induced by ionizing radiation (IR), radiomimetic chemicals, or DNA replication inhibitors. Cells detect and repair DSBs through two distinct but partly overlapping signaling pathways, nonhomologous end joining (NHEJ) and homologous recombination (HR). DNA repair through the HR pathway is restricted to S and G2 phases of the cell cycle, while NHEJ can occur during any cell cycle phase. Defects in both pathways have been associated with human disease, including cancer (1).
DNA repair through the NHEJ pathway involves a core group of proteins that includes the Ku heterodimer, DNA-PKcs, DNA ligase IV, XRCC4, and XLF. XLF interacts with XRCC4 and promotes the ligation of DNA strands by DNA ligase IV and the ligase cofactor XRCC4. The ATP-dependent ligation of free DNA ends is the final step in the NHEJ repair pathway (2). Research studies suggest that XLF and XRCC4 proteins form complexes that bridge DNA breaks earlier in the NHEJ pathway (3). Additional studies indicate that localization of XRCC4 to the nucleus and levels of XRCC4 protein are both regulated by DNA ligase IV (4). Mutations in the corresponding LIG4 gene are associated with LIG4 syndrome, a disorder characterized by immunodeficiency and developmental growth delay. Cells isolated from patients diagnosed with LIG4 syndrome display typical cell cycle checkpoint activity, but aberrant rejoining of DNA double strand breaks (5,6).
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