Proteinase K
目录号 : GP10161
蛋白酶 K (Protease K) 是一种非特异性丝氨酸蛋白酶，可用于蛋白质的一般消化。
Cas No.:39450-01-6
Sample solution is provided at 25 µL, 10mM.
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| Cas No. | 39450-01-6 | M.Wt | 29.3 kDa |
| Specific activity | >30 U/mg | Stability | pH range:4.0 to 12.5, temperature range: 25°C to 65°C. |
| Solubility | H2O : 25 mg/mL (Need ultrasonic) | Storage | Store at -20°C |
| General tips | GlpBio guarantees optimal performance of this product for 18 months after date of delivery under the appropriate temperature and condition. | ||
| Shipping Condition | Evaluation sample solution : ship with blue ice | ||
Quality Control & SDS
- View current batch:
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Specific activity: >30 U/mg
- Datasheet
This protocol is used in the extraction of genomic DNA from mouse tail with Proteinase K.
1. Place an approximately 0.5cm mouse tail into a 1.5 ml microcentrifuge tube.
2. Add 500 μl of lysis buffer (For example: 50 mM KCl, 50 mM Tris-HCl (pH 8.0), 2.5 mM EDTA,0.45% NP-40, 0.45% Tween-20).
3. Add 2.5 μl Proteinase K (20 mg/ml) to the tubeand mixthe solution.
4. Incubate overnight at 55°C.
Optional step:Incubate for an additional 1 hour at 65°C.
5. Vortex the tube and spin down for 10 seconds at 13,000 rpm to collect cell debris.
6. Use 1 μl from the top part of the supernatant per 50 μl of PCR mix(for genotyping).
Proteinase K is a broad-spectrum serine protease and our product is extracted from Pichia pastoris cells with cloned gene encoding Engyodontium album (Tritirachium album) endolytic protease. It is a highly reactive protease frequently used for digesting various proteins and enzymes (including endonuclease, exonuclease, DNase or RNase) .Therefore, it is usually used in DNA preparations without impairing the integrity of the isolated DNA. It has a superior performance under a broad range of conditions: pH, buffer, detergents (such as SDS), chelator (such as EDTA), and temperature. Proteinase K hydrolyzes peptide bonds preferentially adjacent to carboxyl group of hydrophobic amino acids (aliphatic, aromatic, and others).
Except the isolation of genome, it can also take a job in detection of enzyme localization or removal of enzymes from DNA to improve cloning efficiency.
Appropriate working concentration of proteinase K is always among the range of 0.05 to 1 mg/mL. The activity of the enzyme can be stimulated by 0.2 to 1% SDS or by 1 to 4 mol urea. It is activated by calcium (1-5mM), although calcium ions do not affect the enzyme activity, but it contributes to the thermal stability and protects the proteinase from autolysis. Proteinase K has two binding sites for Ca2+, which are located close to the active center, but are not directly involved in the catalytic mechanism. So calcium ion has a regulatory function for the substrate binding site of proteinase K. The enzyme is inactivated by DIFP or PMSF. However, it is not inhibited by EDTA, iodoacetic acid, trypsin-specific inhibitor TLCK, chymotrypsin-specific inhibitor TPCK, and p-chloromercuribenzoate.
We recommend an optimum pH of 7.5 to 8.0 and optimum temperature at 50 to 55°C. Rapid denaturation will occur at temperatures above 65°C. You can hold it under 95°C for 10 min as a heat inactivation.
References:
[1]. Kraus, E; et.al. Proteinase K from the Mold Tritirachium album limber, Specificity and Mode of Action. Z. Physiol. Chem., 357:939; 1976.
[2]. Jany,KD, et al. Amino Acid Sequence of Proteinase K from the Mold, Tritirachium albumlimber. Proteinase K; a Subtilisin-related Enzyme with Disulfide Bonds. FEBS Letter, 199,139.1986.
蛋白酶K是一种广谱的丝氨酸蛋白酶，我们的产品是从克隆Engyodontium album（Tritirachium album）内切蛋白酶基因的毕赤酵母细胞中提取的。它是一种高度反应性的蛋白酶，常用于消化各种蛋白质和酶（包括内切酶、外切酶、DNase或RNase等）。因此，在DNA制备中通常使用它而不会损害所分离的DNA的完整性。它在广泛的条件下具有优越的性能：pH，缓冲液，清洁剂（如SDS），螯合剂（如EDTA）和温度。蛋白酶K首选水疏性氨基酸（脂肪族、芳香族等）的羧基侧链相邻的肽键进行水解。
除了分离基因组外，它还可以在酶定位检测或去除酶从而提高克隆效率方面发挥作用。
适当的蛋白酶K工作浓度始终在0.05至1 mg/mL范围内。酶活性可以通过0.2至1％ SDS或1至4 mol尿素来刺激。它被钙（1-5mM）激活，尽管钙离子不影响酶活性，但它有助于热稳定性并保护蛋白酶不受自溶的影响。蛋白酶K有两个钙离子结合位点，位于活性中心附近，但不直接参与催化机制。因此，钙离子对蛋白酶K的底物结合位点具有调节作用。该酶被DIFP或PMSF失活。但是，它不受EDTA，碘乙酸，胰蛋白酶特异性抑制剂TLCK，胰蛋白酶特异性抑制剂TPCK和对氯汞苯甲酸盐的抑制。
我们推荐最适宜的pH为7.5至8.0，最适宜的温度为50至55°C。在65°C以上的温度下会发生快速变性。您可以将其保持在95°C下进行10分钟的热失活。
| Cas No. | 39450-01-6 | SDF | |
| 分子式 | 分子量 | 29.3 kDa | |
| 溶解度 | H2O : 25 mg/mL (Need ultrasonic) | 储存条件 | Store at -20°C |
| General tips | 请根据产品在不同溶剂中的溶解度选择合适的溶剂配制储备液；一旦配成溶液，请分装保存，避免反复冻融造成的产品失效。 储备液的保存方式和期限：-80°C 储存时，请在 6 个月内使用，-20°C 储存时，请在 1 个月内使用。 为了提高溶解度，请将管子加热至37℃，然后在超声波浴中震荡一段时间。 |
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| Shipping Condition | 评估样品解决方案：配备蓝冰进行发货。所有其他可用尺寸：配备RT，或根据请求配备蓝冰。 | ||
| 制备储备液 | |||
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1 mg | 5 mg | 10 mg |
| 1 mM | 34.1297 mL | 170.6485 mL | 341.2969 mL |
| 5 mM | 6.8259 mL | 34.1297 mL | 68.2594 mL |
| 10 mM | 3.413 mL | 17.0648 mL | 34.1297 mL |
| 第一步：请输入基本实验信息（考虑到实验过程中的损耗，建议多配一只动物的药量） | ||||||||||
| 给药剂量 | mg/kg |  |
动物平均体重 | g | |
每只动物给药体积 | ul | |
动物数量 | 只 |
| 第二步：请输入动物体内配方组成（配方适用于不溶于水的药物；不同批次药物配方比例不同，请联系GLPBIO为您提供正确的澄清溶液配方） | ||||||||||
| % DMSO % % Tween 80 % saline | ||||||||||
| 计算重置 | ||||||||||
计算结果:
工作液浓度： mg/ml；
DMSO母液配制方法： mg 药物溶于 μL DMSO溶液（母液浓度 mg/mL，
体内配方配制方法：取 μL DMSO母液，加入 μL PEG300，混匀澄清后加入μL Tween 80，混匀澄清后加入 μL saline，混匀澄清。
1. 首先保证母液是澄清的；
2.
一定要按照顺序依次将溶剂加入，进行下一步操作之前必须保证上一步操作得到的是澄清的溶液，可采用涡旋、超声或水浴加热等物理方法助溶。
3. 以上所有助溶剂都可在 GlpBio 网站选购。