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Description
Details
Description:Mouse monoclonal antibody to human interleukin 8 (IL-8)
Purification: Protein G affinity purified
Product Type:Primary antibody, detection antibody in matched antibody pair
Target Protein:Human IL-8
Immunogen:Purified recombinant human IL-8
Fusion Myeloma:P3X63Ag8.653
Specificity:This antibody recognizes both recombinant and native human IL-8.
Species Reactivity: Human, others not tested.
Cross-reactivity:This antibody does not cross react with human Monocyte Chemotactic Activating Factor (MCAF) or RANTES (Regulated on Activation, Normal T-cell Expressed, and Secreted).
Host / Isotype: Mouse, IgG1 Kappa
Formulation: Lyophilized in 0.01M PBS, pH 7.0.
Reconstitution:Double distilled water is recommended and to adjust the final concentration to 1.00 mg/mL.
Storage: Store at -20oC
Research Area:Cytokine, interleukin and chemotaxis
Background:
Interleukin 8 (IL-8), like IL-6, is secreted by macrophages and a variety of cells that express Toll-like receptors in response to the stimulation of pathogens. IL-8"s primary function is to recruit neutrophils and other target cells through chemotaxis to the infected site to eliminate pathogens. IL-8 causes increased intracellular Ca2+, release of reactive oxygen species, and other physiological changes required for migration and phagocytosis. IL-8 is also known to promote angiogenesis.
Applications:
1. ELISA: This antibody can be used as detection antibody in sandwich ELISA applications for human IL-8 detection in combination with a monoclonal capture antibody (Cat No: MO-C40017A).
2. Neutralizing: In chemotaxis assay, the antibody inhibited 98.4% of the chemotactic activity of IL-8 on RB/293 cell using 5mg/mL of antibody and 10ng/mL of human IL-8 in assay. This antibody inhibited the chemotactic effect of human IL-8, had no inhibitory effect to MIP 1-bata and RANTES in neutrophil chemotaxis assay, indicating that the antibody’s neutralization activity is specific. The antibody also neutralized IL-8 induced calcium influx in human granulocytes.
3. Western Blot:The antibody used at 0.02-0.1μg/mL concentrations will allow visualization of 100ng/lane of recombinant human IL-8.
Figure: Western blot analysis of recombinant human IL-8 using clone I8-60.
4.IHC: The antibody was used to detect IL-8 expression in the stratum corneum of psoriatic skin tissue. The image below shows the result of detecting with anti-IL-8 clone 60, biotinylated 2rd antibody and avidin-HRP. The specimen was stained with DAB substrate for 5~10 min, and counter-stained with hematoxylin.
References:
If research is published using this product, please inform Anogen in order to cite the reference on this datasheet. Anogen will provide one unit of product in the same category as gratitude.
Additional
Additional Information
| Product Specificity | mAb anti-Human IL-8, I8-60, Detector |
|---|---|
| Application | ELISA, NT, WB, IHC |
| Size | 0.1 mg |
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如果经费充足的话可以找公司包装病毒，如武汉的普健可以提供各种载体的构建及细胞株构建的技术服务。
细胞转染是指将外源分子如DNA，RNA等导入真核细胞的技术。随着分子生物学和细胞生物学研究的不断发展，转染已经成为研究和控制真核细胞基因功能的常规工具。在研究基因功能、调控基因表达、突变分析和蛋白质生产等生物学试验中，其应用越来越广泛。
方法
脂质体转染法
阳离子脂质体表面带正电荷，能与核酸的磷酸根通过静电作用，将DNA分子包裹入内，形成DNA脂复合物，也能被表面带负电的细胞膜吸附，再通过融合或细胞内吞进入细胞。脂质体转染适用于把DNA转染入悬浮或贴壁培养细胞中，是目前实验室最方便的转染方法之一，其转染率较高，优于磷酸钙法。由于脂质体对细胞有一定的毒性，所以转染时间一般不超过24小时。常用细胞类型：cos-7 、BHK、NIH3T3 、Hela等。
电穿孔转染法
电流能够可逆地击穿细胞膜形成瞬时的水通路或膜上小孔促使DNA分子进入胞内，这种方法就是电穿孔。当遇到某些脂质体转染效率很低或儿乎无法转入时建议用电穿孔法转染。一般情况下，高电场强度会杀死50%-70% 的细胞。现在针对细胞死亡开发出了一种电转保护剂，可以大大的降低细胞的死亡率，同时提高电穿孔转染效率。
病毒感染
对于脂质体转染与电穿孔转染都无法成功转染的细胞系建议用病毒感染，此法可以快速100%感染，检测成功率高。
常用步骤
1. 转染试剂的准备
① 将400ul去核酸酶水加入管中，震荡10秒钟，溶解脂状物。
② 震荡后将试剂放在－20摄氏度保存，使用前还需震荡。
2. 选择合适的混合比例(1：1－1：2/脂质体[1]体积：DNA质量)来转染细胞。在一个转染管中加入合适体积的无血清培养基。加入合适质量的MyoD或者EGFP的DNA，震荡后在加入合适体积的转染试剂，再次震荡。
3. 将混合液在室温放置10―15分钟。
4. 吸去培养板中的培养基，用PBS或者无血清培养基清洗一次。
5. 加入混合液，将细胞放回培养箱中培养一个小时。
6. 到时后，根据细胞种类决定是否移除混合液，之后加入完全培养基继续培养24－48小时。
然后你得确定，这个细胞株对于你得研究来说不可或缺，具有明确的代表性。
上面两个都没有问题的话，祝你顺利。
① 在构建载体时，目的基因直接整合到细胞染色体组上，最好不要通过先瞬转在筛选稳定细胞株的这种方法，因为转染效率没有保证
② 高表达载体的构建，哺乳动物表达量一直是它自身的缺点，最好根据高表达载体定向的驯化细胞，提高蛋白表达量
③ 细胞的选择，筛选稳定细胞株我们常用的细胞是CHO，中国仓鼠卵巢细胞，由于CHO具有诸多的优点因此适合用于筛选稳定细胞株，而HEK293细胞则常用于瞬时转染
④ 后期的筛选，双抗预防污染，筛选细胞的时候抗生素浓度一定要做预实验，而且转染的时候不能有抗生素，关于细胞转染 稳定细胞系构建的相关理论
