Assay Method Information | |
| DNA Helicase Assay |
Description: | Helicase assay was demonstrated using the purified fraction of PfUDN. The specially designed partial duplex substrate consisted of a 32P-labelled 47-mer DNA oligodeoxynucleotide annealed to M13mp19 phage ssDNA. This oligodeoxynucleotide of the nucleotide sequence 5'-(T)15GTTTTCCCAGTCACGAC(T)15-3' contains 15 base-pairs of non-complementary region (T)15 at both the 5' and 3' ends. Oligodeoxynucleotide was labeled at 5'-end with T4 polynucleotide kinase (PNK) (5U) (New England Biolabs) and 1.85 MBq of [γ-32P] ATP (specific activity 222 TBq/mmol) at 37°C for one hour and then annealed using standard annealing buffer (20 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 100 mM NaCl, 1 mM DTT) with 0.5 μg of single-stranded circular M13mp19 (+)phage DNA by heating at 95°C for 1 min and then transferring immediately to 65°C for 2 min and then slow cooling to room temperature. Using gel filtration through a Sepharose 4B column (Pharmacia, Sweden) the nonhybridized oligodeoxynucleotide was removed [Ahmad et al., PLoS One, 7(11):e49385]. The reaction volume of 10 μl containing the 32P-labeled helicase substrate (1000 cpm/10 μl) in appropriate buffer (20 mM Tris-HCl, pH 8.0, 8 mM DTT, 1.0 mM MgCl2, 20 mMKCl and 16 μg/ml BSA) and PfUDN was incubated at 37°C for 60 min. The substrate and products were separated by electrophoresis on a nondenaturing 12% PAGE and the gel was exposed to hyper film for autoradiography or scanned on phosphoimager. |
Affinity data for this assay | |
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