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Human DNA polymerase beta - for 100 assays

BHE16300228

This product includes 7 ul of 10 uM human DNA polymerase beta (1000 x stock).

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orders@biohippo.com
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Reference 1. Merlo S., et al, Identification of 5‑Methoxyflavone as a Novel DNA Polymerase-Beta Inhibitor and Neuroprotective Agent against Beta-Amyloid Toxicity. J. Nat. Prod. 78, 2704−2711 (2015). 
2. Maldonado E. et al, Expression, purification, and biochemical characterization of recombinant DNA polymerase beta of the Trypanosoma cruzi TcI lineage: requirement of additional factors and detection of phosphorylation of the native form, Parasitol Res (2015) 114:1313–1326.  
Product Manual Datasheet
Protocols Description Assay Protocol using Human DNA Polymerase Assay kit (Catalog No. DPB100K)
1. Reagent preparation:
DNA, dTNP mix and dye are diluted with water. The DNA polymerase is diluted with 1 x assay buffer.

2. Reaction:
The total volume of each reaction mixture is 50µl including: 30µl of H2O, 5µl of 10 x buffer (Buffer PB), 5µl of 10 x DNA, 5µl of 10 x human DNA polymerase beta, 5µl of 10 x dNTP mix. Incubate the reaction mixture in a standard black 96-well plate at 37°C for 60 min.
 
Note: The final concentrations in the assay reaction are 30 mM Tris-HCl, pH 8.0, 10 mM MgCl2, 1 mM DTT, 5 % glycerol, 150 nM DNA, 0.1 mM dATP, 0.1 mM dGTP. The recommended final enzyme concentration in the reaction mixture is 10 U/ml or 10 nM. The diluted enzyme should be prepared freshly.
 
3. Detection:
For each reaction, make 240µl of detection solution by mixing 200µl of Reagent U and 40µl of the 1 x fluorescence dye. Mix 240µl of detection solution with the 50µl of reaction mixture. Measure the fluorescence intensity at 535 nm using the excitation wavelength at 485 nm.
 
Note: The fluorescence signal decreases in the first 30 min after addition of the detection solution. For quick tests, the fluorescence can be measured right after addition of the detection solution or at a fixed time point, for example, 5 min after addition of the detection solution. For HTS applications, where many plates may be accumulated and the reading time of different plates may vary, it is recommended reading the plates in 30 min or a longer period of time after addition of the detection solution.
Combination SKU Supplier No. Size Your price Quantity
BHE16300228-1 HDPB-100 100 assays $421.56
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BHE16300228-2 HDPB-1000 1000 assays $2950.91
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