The Tdp1 Assay Kit contains all reagents for routine detection of Tdp1 activity using the tyrosine release assay from the substrate Oligo-Tyr (human tdp1 is sold separately). The assay is highly specific for Tdp1.
Volumes are given for the 100 assay kit size. For the 250 assay kit size, multiply volumes by 2.5 fold. Note that this kit does not include Tyrosyl DNA Phosphodiesterase, which is sold separately (see link above). We recommend reconstructing the assay using the purified Tdp1 to ensure that the assay is optimized and working well.
- Oligo-Tyr substrate (10 ul at 0.8 ug/ul in TE buffer [10 mM Tris, pH 7.5, 1 mM EDTA]). This substrate should be end labeled with T4 polynucleotide kinase and g-[32P]- ATP as described in the kit protocol
- 5x Tdp1 assay buffer (300 ul): 5x buffer contains 100 mM Tris-HCl (pH 7.5), 500 mM KCl, 50 mM EDTA, 5mM dithiothreitol (DTT).
- 2x Stop Buffer (gel loading buffer) (200 ul): 96% formamide, 20 mM EDTA, 0.03% xylene cyanol, 0.03% bromophenol blue.
This kit may shipped on wet ice (or dry ice internationally). The buffers and oligo can be stored at 4°C (short term storage) or at -20°C (long term). The components in this kit are not sensitive to freezing and thawing.
Assay Protocol Overview:
Reaction volumes should be 10-20 ul final volume (limited by volume that can be loaded onto the gel). Reactions are assembled in microfuge tubes with water, buffer and Oligo-Tyrosine substrate. The test fractions should be added last and the reactions incubated at 37 C for at least 15 min. then terminated with 10 ul of the stop buffer. Analyze on a denaturing polyacrylamide gel or by DNA sequencing gel electrophoresis.
Actual Tdp1 assay is shown below. In this analysis, 5’ end labeled Oligo, linked to a tyrosine moiety on the opposite (3’) end, was incubated with purified human Tdp1 from TopoGEN. The release of tyrosine was measured on a DNA sequencing gel that monitors a change in molecular weight.