| Contents: |
4140-100-01 DNA Ligase IV/XRCC4 Tetramer 100 units
3900-500-13 10X REC Buffer 13 1 ml
4140-100-02 1X Storage/Dilution Buffer 1 ml
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| Source: |
Eukaryotic expression system coexpressing recombinant human DNA Ligase IV and XRCC4 |
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| Unit Definition: |
One unit is the amount of enzyme required to ligate oligo (dT) 24 when hybridized topoly (dA)300 at the rate of 1 pmole in 30 minutes at 37°C.
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| Assay Conditions: |
Reactions of 20 µl contain 4 pmoles of 32P-oligo(dT)24 , 0.2 pmole of poly(dA)300 , 1X REC™ Buffer 13 (50 mM Tris-Cl (pH 7.5), 10 mM MgCl 2 , 100 μg/ml BSA, 10 mM DTT, 1 mM ATP, 0.1% Triton X-100), and serial dilutions of DNA Ligase IV/XRCC4 are incubated at 37°C for 30 minutes. Ligation products are resolved by 20% denaturing polyacrylamide gel electrophoresis, detected by autradiography, and quantitated.
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| Applications: |
Studies involving NHEJ
Duplex ligation assays
Irradiation sensitivity assays
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| Storage: |
The enzyme may be diluted in 1X Storage/Dilution Buffer (Cat# 4140-100-02)and stored frozen in working aliquots at -80 . C. Avoid repeated freeze-thaws. |
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| Reference: |
1. Lee, K.J., Huang, J., Takeda, Y., Dynan, W.S. 2000. DNA ligase IV and XRCC4 form a mixed tetramer that functions synergistically with other repair factors in a cell-free end-joining assay. J Biol Chem 275:34787-34796.
2. Chen, L., Trujillo, K., Sung, P., Tomkinson, A.E. 2000. Interactions of the DNA ligase IV-XRCC4 complex with DNA ends and the DNA-dependent protein kinase. J Biol Chem 275:26196-26205.
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