Wako 125-05061 赖氨酰肽链内切酶使用说明书
质谱级赖氨酰肽链内切酶 Lysyl Endopeptidase
【产品详情】
赖氨酰肽链内切酶
品牌:WAKO
级别:质谱级
赖氨酰肽链内切酶Lysyl Endopeptidase 125-05061 wako
英文品名:Lysyl Endopeptidase?, Mass Spectrometry Grade
货号:125-05061
规格:5×20 μg
应用:蛋白质组学。
用途:赖氨酰肽链内切酶,蛋白测序、质谱分析,多肽合成
赖氨酰肽链内切酶Lysyl Endopeptidase 125-05061 wako
实验方法
1. 试剂:
A.0.2 mol/L AMP 缓冲液,pH值 9.5
溶解 4.2 g 的2-氨基-2-甲基-1,3-丙二醇于 150 mL 的水中,加入1 mol/L HCl 调 pH 值至 9.5,再加水使体积至 200 mL。
B.2.5 mmol/L 底物溶液
溶解 22.6 mg 的N-苯甲酰基-DL-精氨酰-4-硝基苯胺盐酸盐于 20 mL 水中。
C. 2 mmol/L Tris-HCl 缓冲液,pH8
溶解 0.24 mg的2-氨基-2-羟甲基-1,3-丙二醇于 900 mL 水中,加入 0.1 mol/L HCl 调pH值至8,再加水使体积至1 L。
D. 酶溶液
溶解1vial 的赖氨酰肽链内切酶于1mL 的溶剂C中,可直接加入。
E. 终止溶液
将 55 mL 水和 45 mL 乙酸混合均匀。
2. 步骤
试剂 | 检测样品 | 空白对照 |
A | 2.6 mL | 2.6 mL |
B | 0.3 mL | 0.3 mL |
30℃ 预培养5分钟 | ||
D | 0.1 mL | – |
C | – | 0.1 mL |
立即混合均匀,30℃ 预培养25分钟 | ||
E | 1.0 mL | 1.0 mL |
3. 单位的定义
酰胺酶单位是指 30℃、pH 9.5 时,每分钟产生1μmol 对硝基苯胺的酶量。
AU/vial = [(a-b) / 25] × (1/9.62) × (4.0/0.1)
a. 检测样品中的吸光度
b. 空白对照中的吸光度
胶内酶切的实验操作流程
用聚硅酮处理的微量离心管和吸管端防止捕获任何蛋白。使用质谱分析用凝胶染色试剂盒,例如银染剂 MS 试剂盒(产品编号:299-58901)和负凝胶染色 MS 试剂盒(产品编号:293-57701)
1. 电泳分离蛋白质样品;
2. 从凝胶中切割蛋白质片断并放入微量离心管;
3. 使凝胶脱色(可使用质谱分析用凝胶染色试剂盒中的脱色溶液);
4. 加入300 μL 乙腈到试管里,搅拌器振荡 30 分钟;
5. 去除乙腈,用 Parafilm 膜覆盖微量离心管。
6. 在 Parafilm 膜上打出针孔,真空干燥 15 分钟;
7. 100 μL 10 mmol/L DTT 溶解于 100 mmol/L 碳酸氢铵,56℃恒温1小时。
8. 室温冷却后,用等量的 50 mM 碘乙酰胺溶解于 100 mmol/L 碳酸氢铵,暗处恒温 45 分钟并涡旋;
9. 用 100 μL 100 mmol/L 碳酸氢铵洗涤凝胶片段 10 分钟;
10. 用 300 μL 乙腈干燥凝胶片段 15 分钟;
11. 用 100 μL 100 mmol/L 碳酸氢铵溶胀凝胶片段 15 分钟;
12. 用 300 μL 乙腈再次干燥凝胶片段 15 分钟;
13. 去除液相,真空干燥凝胶片段 15 分钟;
14. 用 100 μL 赖氨酸内切酶溶液*在冰水浴中溶胀凝胶片段 45 分钟;
*赖氨酸内切酶稀释于 50 mmol/L Tris-HCl pH 8.5;
15. 去除 100 μL 赖氨酸内切酶溶液,将凝胶片段放在 37℃、10 μL 50 mmol/L Tris-HCl pH 8.5 中过夜;
16. 加入 50 μL 20mmol/L 碳酸氢铵 20 分钟内振荡凝胶片段3次抽提多肽;
17. 加入 5% 甲酸/50% 乙腈 20 分钟内振荡凝胶片段3次抽提多肽;
18. 如果需要用 Speed Vac. 浓缩多肽;
19. 用 ZipTip 脱盐和纯化多肽;
20. 如果需要用弱真空浓缩多肽至2 μL;
21. 加入基质进行质谱分析。
注意:根据细菌的生理和形态特征分类,产品来源为水解无色杆菌,但是最近细菌分类学将这种细菌鉴定为产酶溶杆菌。
保存:暗处-20℃保存
规格:20 μg×5 vial
Wako 125-05061 赖氨酰肽链内切酶使用说明书 参考文献
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产品编号 | 产品名称 | 产品规格 | 产品等级 |
125-05061 | Lysyl Endopeptidase®, MS Grade 赖氨酰肽链内切酶,MS级 |
20 μg×5 | 质谱级 |
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