| 货号 (SKU) | 包装规格 | 是否现货 | 价格 | 数量 |
|---|---|---|---|---|
| H418757-250U | 250U |
期货  |
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| H418757-500U | 500U |
期货 |
| |
| H418757-1000U | 1000U |
期货 |
| |
| H418757-2000U | 2000U |
期货 |
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| 英文名称 | Human Topoisomerase IIα |
|---|---|
| 储存温度 | 避免反复冻融,-80℃储存 |
| 运输条件 | 超低温冰袋运输 |
| 产品介绍 |
产品信息 拓扑异构酶 II 测定条件 在 topo II 反应缓冲液（50 mM Tris-Cl, pH 8.0, 120 mM KCl, 10 mM MgCl2. 0.5 mM ATP, 0.5 mM 二硫苏糖醇）中使用最终体积为 20-30 μl 的动质体 DNA 底物与0.2 μg KDNA。用 5 μl（每 20 μl 反应体积）终止缓冲液（5% 肌氨酸、0.0025% 溴酚蓝、25% 甘油）终止反应。在含有 0.5 ug/ml 溴化乙锭的 1% 琼脂糖凝胶上分析反应产物。在凝胶运行时，可以使用手持式紫外光源轻松监测 2.5 kb DNA 小环去链产物的分辨率。 描述 纯化的酶完全不含拓扑异构酶 I 污染和核酸酶。它以两步改变独特拓扑异构体的连接数，在SDS-PAGE上检测到的主要多肽为170 kDa；在过载的凝胶上看不到其他条带。 拓扑异构酶 II 质量控制测试 核酸酶污染：通过测试线性 kDNA 和线性质粒 DNA 形成进行测定。将 1 μg 链状 KDNA 或超螺旋 pUC19 DNA 孵育 4 小时。在 37°C（在 topo II 测定条件下，有或没有 ATP）。 通过在抑制 topo II 活性的条件下测定超螺旋 DNA 的松弛来评估与 topo I 的交叉污染。在没有 ATP（有和没有 Mg++）的情况下，过量的纯化酶与 pRYG DNA 在 37°C 下孵育 2 小时。在这些条件下，不得检测到 pRYG 超螺旋 DNA 的松弛。 当 SDS-PAGE 凝胶被纯蛋白质超载时，可以看到一条 170 kDa 的单条带。最后一部分中没有 180 kDa 拓扑 II 形式。（蛋白质浓度因批次而异，但通常在 5-50 ug/ml 的范围内。） Topo IIa 的供应量为 2-10 单位/ul（1 单位将在 37 分钟内 30 分钟内释放 0.2 ug KDNA ℃）。 topo IIa 的最后一部分在以下缓冲液中从 FPLC 柱中脱落：10% 甘油、50 mM Tris (pH 7.7)、1 mM EDTA 和 EGTA、650 mM NaCl。 运输和储存 酶在干冰上运输，应储存在 -70°C。我们还建议在第一次解冻后对酶进行分装（反复冻融会导致活性丧失）；酶活性稳定1-2天（不长）。 Product Info Topoisomerase II Assay Conditions Decatenation assays are carried out using kinetoplast DNA substrate in a final volume of 20-30 μl in topo II reaction buffer (50 mM Tris-Cl, pH 8.0, 120 mM KCl, 10 mM MgCl2. 0.5 mM ATP, 0.5 mM dithiothreitol) with 0.2 μg KDNA.? Reactions are terminated with 5 μl (per 20 μl reaction volume) of stop buffer (5% sarkosyl, 0.0025% bromophenol blue, 25% glycerol). Reaction products are analyzed on a 1% agarose gel containing 0.5 ug/ml ethidium bromide.? Resolution of a 2.5 kb DNA minicircle decatenated product can be easily monitored with a hand held UV light source while the gel is running. Description The purified enzyme is completely free of topoisomerase I contamination and nucleases.? It alters linking number of a unique topoisomer in steps of two and the major polypeptide detected on SDS-PAGE is 170 kDa; no other bands are visible on an overloaded gel. Topoisomerase II Quality Control Tests Nuclease contamination: assayed by testing for linear kDNA and linear plasmid DNA formation. Incubation of 1 μg of catenated KDNA or supercoiled pUC19 DNA for 4 hrs. at 37° C (under topo II assay conditions and with or without ATP). Cross contamination with topo I was assessed by assaying for relaxation of supercoiled DNA under conditions where topo II activity is suppressed.? Excess purified enzyme was incubated with pRYG DNA in the absence of ATP (with and without Mg++) for 2 hr at 37° C.? Under these conditions, relaxation of pRYG supercoiled DNA must not be detected. A single band of 170 kDa was seen when an SDS-PAGE gel was overloaded with the pure protein. There is no 180 kDa topo II form in the final fraction. (Protein concentration is variable from lot to lot but usually is in the range of 5-50 ug per ml.) Topo IIa may be supplied at 2-10 units/ul (1 unit will decatenate 0.2 ug of KDNA in 30 min at 37°C). The final fraction of topo IIa comes off an FPLC column in the following buffer: 10% glycerol, 50 mM Tris (pH 7.7), 1 mM EDTA and EGTA, 650 mM NaCl. Shipping&storage The enzyme is shipped on dry ice and should be stored at -70°C. We also recommend that the enzyme be aliquoted after the first thaw (repeated rounds of freeze/thaw will lead to loss of activity); the enzyme activity is stable for 1-2 days (not longer). |
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