Sequencing gel loading dye

The 5′-kinase activities of wild-type and mutant PNKP proteins were measured by a kinase assay modified from procedures described before^{15 (link)}. Briefly, PNKP (500 ng) was added to a reaction mixture (40 µL total volume) containing kinase buffer (80 mM succinic acid, 10 mM MgCl₂ and 1 mM DTT, pH 5.5), 0.5 mM 24-mer oligonucleotide substrate (5′-GGCGCCCACCACCACTAGCTGGCC-3′) with 5′-OH termini, 5 mM unlabeled ATP, and 5 µCi of [γ-³²P] ATP (3000 Ci/mmol, Amersham Pharmacia Biotech). The reaction mixture was incubated at 37 °C for 0.5, 1, 2, 5, 10 and 20 min. 5 µL of the sample was mixed with 2.5 µL of 3 × sequencing gel loading dye (Fisher Scientific, Edmonton, AB), boiled for 10 min to stop the reactions then run on a 12% polyacrylamide gel containing 7 M urea at 200 V for 30 min. The gel was scanned on a Typhoon 9400 Variable mode imager (GE Healthcare Life Sciences, Mississauga, ON) and quantified using ImageQuant 5.2 (GE Healthcare Life Sciences). In the experiment to test the stability of the mutant proteins in vitro, all the purified wild-type/mutant PNKP proteins were pre-heated at 37 °C for 10 min before being added into the reaction mixture.

PNKP (10 pmol) was premixed with 40 pmol of full-length XRCC1-His, His-XRCC1^FFF, His-XRCC1^161–406, or His-XRCC1^{161–406 R335A/K369A} at 37 °C for 5 min and then the mixtures were added to 20-μl (total volume) reactions containing kinase buffer (80 mm succinic acid, pH 5.5, 10 mm MgCl₂, and 1 mm dithiothreitol), 0.2 nmol of 24-mer 5′-DNA kinase substrate (Integrated DNA Technologies; the single- and double-stranded DNA substrates used in this study have been described previously (32 (link))) and 3.3 pmol of [γ-³²P]ATP (PerkinElmer Life Sciences) and incubated for 2 min at 37 °C. 4-μl aliquots were mixed with 2 μl of 3× sequencing gel loading dye (Fisher), boiled for 10 min, and fractionated on a 12% polyacrylamide, 7 m urea sequencing gel at 200 V. Gels were imaged on a Typhoon 9400 variable mode imager (GE Healthcare, Bucks, UK) and quantified using ImageQuant 5.2 software (GE Healthcare).