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Home / Enzymes, Inhibitors & Substrates / Human Enzymes

Human Topoisomerase I

SKU : BHE16300230

This product includes 20 ul of 2,000 U/ul of purified human Topoisomerase I form sf9 cells and 4 ml of 10 x assay buffer.

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ProFoldin

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Mfr.No.	HTOPI-040, HTOPI-010, HTOPI-100
Description	Protein construct: Wild-type human topoisomerase I purified from a baculovirus insect protein expression system. MW: 92 kDa, single subunit Enzyme concentration: 500 U / µl for HTOPI-010; 2000 U / µl for HTOPI-040 and HTOPI-100. Enzyme Unit: One unit is the enzyme amount that relaxes 0.25 μg of supercoiled plasmid DNA in 30 minutes at 37 °C. DNA Relaxation assay: Use 4 U of Topoisomerase I and 1µg of supercoiled DNA for DNA relaxation assays at 37°C. Use a higher enzyme concentration if the reaction temperature is room temperature. The DNA relaxation activity is detected by agarose gel electrophoresis or 96-well plate fluorescence assay. Human DNA relaxation assay kit is available from ProFoldin (Catalog No. HRA100K for 100 assays). Reagents for HTS quantities are available. Storage temperature: 4°C for short term or -80°C for long term storage. Do not freeze-and-thaw repeatedly. Enzyme dilution: Use the 1 x Buffer HT1 to dilute the enzyme just before the assay. Do not store diluted enzyme solution. Human Topoisomerase I, 40,000 Units (Catalog No. HTOPI-040) contains 20 µl of 2,000 U/µl of purified human topoisomerase I.

Product Manual	Datasheet
Protocols Description	DNA Relaxation Assay Protocol using the Human Topoisomerase I DNA Relaxation Assay Kit 1. Reaction and sample preparation: The total volume of each reaction mixture is 40 µl including: 28 µl of H2O, 4 µl of 10 x Buffer HT1, 4 µl of 10 x supercoiled DNA (250 µg/ml), 4 µl of 10 x enzyme (1000 U/ml). Incubate the reaction mixture at 37°C for 60 min. Note: The final concentrations are 25 mM Tris-HCl, pH 7.4, 58 mM KCl, 0.25 mM DTT, 5 mM MgCl2, 0.25 mM EDTA, 15 µg/ml BSA, 25 µg/ml supercoiled plasmid DNA and 100 U/ml topoisomerase I. A negative control reaction can be the reaction mixture without the enzyme. 2. Assay (1) For each 10 assays, freshly dilute 2 µl of 1500 x H19 dye into 3 ml of 1x H19 dilution buffer. The 1x H19 dilution buffer is prepared by 10 fold dilution of the 10x H19 dilution buffer with water. (2) Mix 250 µl of the freshly prepared 1 x H19 dye with each reaction solution (40 µl). Incubate the mixture at room temperature for 5 min. (3) Measure the fluorescence intensity at 535 nm using the excitation wavelength at 485 nm.

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Combination SKU	Supplier No.	Size	Your price	Quantity
BHE16300230-1	HTOPI-040	40000 Units	$1132.86		Add to Cart
BHE16300230-2	HTOPI-010	10000 Units	$606.89		Add to Cart
BHE16300230-3	HTOPI-100	100000 Units	$2623.98		Add to Cart

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