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- 详细信息
- 文献和实验
- 技术资料
- 库存:
56
- 英文名:
Recombinant Human OAS1 protein
- 保质期:
12个月
- 供应商:
上海帛科
- 保存条件:
-20 to -80 °C
- 规格:
50μg、100ug 、1mg
| 货号 | BK-DB5057 | 用途 | 仅供科研研究实验 |
| 纯度 | >90% as determined by SDS-PAGE | 预测分子量 | 36.07 kDa |
| 表达宿主 | E.coli | 种属 | Homo sapiens (Human) |
蛋白构建: A DNA sequence encoding the human OAS1 (Ser74-Pro165) was fused with GST tag
制剂: Supplied as solution form in PBS pH 7.5 or lyophilized from PBS pH 7.5.
运输方式: In general, proteins are provided as lyophilized powder/frozen liquid. They are shipped out with dry ice/blue ice unless customers require otherwise.
稳定性&储存: Use a manual defrost freezer and avoid repeated freeze thaw cycles.Store at 2 to 8 °C for one week .Store at -20 to -80 °C for twelve months from the date of receipt.
复溶: Reconstitute in sterile water for a stock solution.A copy of datasheet will be provided with the products, please refer to it for details.
分子别名: 2'-5'-oligoadenylate synthase 1,(2-5')oligo(A) synthase 1,2-5A synthase 1,E18/E16,p46/p42 OAS.
背景介绍: Interferon-induced, dsRNA-activated antiviral enzyme which plays a critical role in cellular innate antiviral response. In addition, it may also play a role in other cellular processes such as apoptosis, cell growth, differentiation and gene regulation. Synthesizes higher oligomers of 2'-5'-oligoadenylates (2-5A) from ATP which then bind to the inactive monomeric form of ribonuclease L (RNase L) leading to its dimerization and susequent activation. Activation of RNase L leads to degradation of cellular as well as viral RNA, resulting in the inhibition of protein synthesis, thus terminating viral replication. Can mediate the antiviral effect via the classical RNase L-dependent pathway or an alternative antiviral pathway independent of RNase L. The secreted form displays antiviral effect against vesicular stomatitis virus (VSV), herpes simplex virus type 2 (HSV-2), and encephalomyocarditis virus (EMCV) and stimulates the alternative antiviral pathway independent of RNase L.
重组蛋白质的可溶性鉴定:
1. 将按上法诱导培养后收集的菌体重悬于裂解液 1 (Lysis buffer under native conditions )中,然后在-80 oC 低温冰箱中放置 10 min。
2. 冰中解冻。
3. 在冰浴上用超声破碎仪破菌 6 次, 每次 10 sec,间歇 10 sec,电压 200-300 V。
4. 10000g,4oC,离心 20min,取上清(为溶液 A), - 20 oC 保存; 另将沉淀用 同样裂解液 1 溶解(为溶液 B),同样 -20 oC 保存,供后继分析使用。
5. 将上述 A、B 溶液和诱导前后的细菌进行 SDS-PAGE 电泳, 考马斯亮蓝染色, 比较分析重组蛋白质的溶解性。如果诱导表达的蛋白质位于 A 溶液中, 则为可溶性蛋白;如果是在 B 溶液中,则为非可溶蛋白。
重组蛋白质的分离纯化:
1. 将菌体沉淀溶于适量裂解液 2(Lysis buffer under denaturing conditions )中,室温下搅拌和吹打沉淀,避免泡沫生成。
2. 10000g ,4 oC,离心 30 min,收集上清液。
3. 将 Ni-NTAAgarose 充填柱子,并连接于 Pharmarcia 低压液相层析系统,用 5 倍柱体积的裂解液 2 平衡 Ni-NTAAgarose,调节 A280 值至零线。
4. 将适量上清液上样到 Ni-NTAAgarose 柱子中, 并用 lysis buffer 冲洗至 A280 值低于 0.01。
5. 分别用 5 ~ 10 倍柱体积的清洗液 1 和清洗液 2(Washbuffer 1 and 2)清洗柱 子,直至 A280 值低于 0.01。
6. 用洗脱液(Elution buffer)洗脱重组蛋白质, 在 A280 值监测下,收集出现峰 线后含有重组蛋白的所有洗脱液。
表达载体在基因工程有以下几种元件:
(1)选择标志的编码序列;
(2)可控转录的启动子;
(3)转录调控序列(转录终止子,核糖体结合位点);
(4)一个多限制酶切位点接头;
(5)宿主体内自主复制的序列。
重组蛋白质的诱导表达:
1. 挑取转化有质粒的单菌落, 接种于 3ml 选择性 LB 液体培养基中, 37 oC,250 rpm/min 振摇培养过夜。
2. 次日将培养过夜的菌液 500 μl 再接种于 10ml(1:20)选择性 LB 液体培养基 中, 37 oC,250 rpm/min 振摇培养至光密度(OD600=0.6)时,取 1 ml 样本作 为诱导前标本, 10000g 离心 1min 收集菌体沉淀,-20 oC 冻存备用。
3. 加入 1 mol/L IPTG 于菌液中,使 IPTG 终浓度为 1 mM ,37 oC ,250 rpm/min 振摇培养 4 ~ 5 小时。取 1 ml 样本作为诱导后标本,同上法收集菌体沉淀, -20 oC 冻存备用。
4. 将诱导前后菌体沉淀用 20 ~ 40 μl PBS(pH= 8.0)重悬, 加入等体积的 2×SDS 上样缓冲液, 煮沸加热 5min,SDS 聚凝胶(SDS-PAGE)电泳分离 , 考马斯亮蓝染色 3 小时后,脱色观察结果。
5. 选取诱导成功的细菌克隆,扩大诱导规模,收集菌体沉淀,于- 20 oC 保存,准备做下一步分析及纯化。
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