APE1 endonuclease activity was monitored using purified recombinant APE1. Enzymatic reactions were carried out in a final volume of 10 μl using 2.3 fmol of protein in a buffer containing 50 mM HEPES pH 7.5, 50 mM KCl, 10 mM MgCl2, BSA 1μg/μl and 1 mM DTT; samples were pre-incubated for 15 minutes at 37°C with 230 pmol of the selected inhibitors. Reactions were started by adding 100 nM of double stranded abasic DNA substrate (obtained by annealing a DY-782-labeled oligonucleotide 5′-CTTGGAACTGGATGTCGGCACFAGCGGATACAGGAGCA-3′ (Dyomics), where F indicates a tetrahydrofuran residue, with the complementary sequence 5′-TGCTCCTGTATCCGCTGTGCCGACATCCAGTTCCAAG-3′) and incubated at 37°C for the indicated time points. Reactions were halted by addition of formamide buffer (96% formamide, 10 mM EDTA and gel Loading Buffer 6× (Fermentas)), separated onto a 20% (w/v) denaturing polyacrylamide gel and analyzed on an Odyssey CLx scanner (Li-Cor Biosciences). The percentage of substrate converted to product was determined using the ImageStudio software (Li-Cor Biosciences).
Gel loading buffer 6
Manufactured by Thermo Fisher Scientific
Gel Loading Buffer 6x is a concentrated solution designed for preparing DNA or RNA samples prior to loading them into an agarose or polyacrylamide gel for electrophoresis. It contains dyes and glycerol to facilitate sample tracking and loading into the gel.
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2 protocols using gel loading buffer 6
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Monitoring APE1 Endonuclease Activity
2
Quantification of APE1 Endonuclease Activity
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APE1 endonuclease activity was monitored using HeLa cell extracts as follows:13 (link), 34 (link). Briefly, enzymatic reactions were carried out in a final volume of 10 µl using 12.5 ng of cell extracts in a buffer containing 50 mM Tris-HCl pH 7.5, 50 mM KCl, 10 mM MgCl2, BSA (1 μg ml−1), and 1 mM DTT. Extracts were incubated for 15 min, at 37 °C, with 100 nM of double-stranded 26-mer abasic DNA substrate containing a single tetrahydrofuranyl artificial AP site at position 14, which is cleaved to a 14-mer in the presence of AP endonuclease activity34 (link). The double-strand DNA was obtained by annealing a 5ʹ-DY-782-labeled oligonucleotide 5ʹ-AATTCACCGGTACCFTCTAGAATTCG-3ʹ (where F indicates the tetrahydrofuran residue), with an unlabeled complementary sequence 5ʹ-CGAATTCTAGAGGGTACCGGTGAATT-3ʹ.
Reactions were halted by addition of formamide buffer (96% formamide, 10 mM EDTA and gel Loading Buffer 6× (Fermentas)), separated onto a 20% (w/v) denaturing polyacrylamide gel and analyzed on an Odyssey CLx scanner (Li-Cor Biosciences). The percentage of substrate converted to product was determined using the ImageStudio software (Li-Cor Biosciences).
Reactions were halted by addition of formamide buffer (96% formamide, 10 mM EDTA and gel Loading Buffer 6× (Fermentas)), separated onto a 20% (w/v) denaturing polyacrylamide gel and analyzed on an Odyssey CLx scanner (Li-Cor Biosciences). The percentage of substrate converted to product was determined using the ImageStudio software (Li-Cor Biosciences).
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