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Description
Details
Description:Mouse monoclonal antibody to human Transforming Growth Factor beta (TGF-β)
Purification: Protein G affinity purified
Product Type:Primary antibody
Target Protein:Human TGF-β
Immunogen:TGF-βfrom human platelets
Fusion Myeloma: Sp2/0-Ag14
Specificity:Western blotting demonstrated that this antibody reacts with the dimeric (25 kDa) and monomeric (12.5 kDa.) forms of TGF-β under both non-reducing and reducing conditions respectively. This antibody recognizes both human platelet-derived and recombinant TGF-β in ELISA.
Species Reactivity: Human, others not tested.
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 1.00mg/mL.
Storage: Store at -20oC
Research Area:Growth Factors and Their Receptors, Angiogenesis
Background:
Transforming growth factor beta (TGF-β) has three isoforms (TGF-β1, TGF-β2, and TGF-β3) with similar functions.
The cytokine is a homodimer linked by disulfide bind. Inside cells, the cytokine forms a small latent complex with latent associated peptide (LAP). This small complex binds to latent TGF-β binding protein (LTBP) to be secreted to extra-cellular matrix. Disassociation of the latent proteins from TGF-β results in the release of the cytokine to its receptor. The process is called activation, which can be influenced by various factors, including proteases, metalloproteases, extreme pH, mild acidic condition, reactive oxygen species and integrins.
TGF-β is an anti-proliferation factor in normal cells. It increases the synthesis of p15 and p21, which can block the cyclin: CDK complex, and causes cells to stop at G1 phase. The cytokine can induce apoptosis through both SMAD and DAXX pathways. In cancer cells, TGF-β signaling is altered and TGF-β no longer stops cell proliferation.
Applications:
ELISA: The antibody reacts with TGF-β.
Western Blot: This antibody when used at concentration of 5-20ng/mL will allow visualization of 100ng/lane of TGF-β.
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 TGF-β, 2C5 |
|---|---|
| Application | EIA, WB |
| Size | 0.1 mg |
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对于瞬时转染的检测：如果有报告基因，就通过报告基因的表达看转染成功与否；没有报告基因的话，就在24～96h内收获细胞通过RT-PCR和WB来鉴定。
1.将外源基因插入慢病毒载体
2.将构建完成的载体与慢病毒包装质粒混合，共转染靶细胞
3.收集病毒液
4.用病毒液感染靶细胞
5.用载体上带的抗生素进行筛选，如果没有，可以用无限稀释法
6.获得稳转株
一种是脂质体转染后,单克隆筛选稳定细胞株.另外一种是应用逆转录病毒,慢病毒转染,筛选稳定细胞株.
脂质体转染：在转染24小时后,消化细胞并计数.将细胞种到96孔板,保证每个孔2-3个细胞,这样才能得到单克隆.待细胞贴壁后,加入抗生素筛选.筛选时间和浓度视细胞而定.一般G418一个星期作用,嘌呤霉素2-3天.
脂质体法筛单克隆时间较长,且效率低,大概只有1%.
病毒转染：先要用包装细胞,一般为293细胞,包装出病毒,再用病毒转染目的细胞.包装病毒视不同类型的病毒而定,一般要3-5天的时间.包装好的病毒要测滴度,根据滴度决定转染目的细胞的病毒量.转染目的细胞1-2天后加抗生素筛选得到稳定细胞株.
病毒转染得到稳定细胞株的效率高,只是步骤繁琐.
推荐了解“主细胞库”“工作细胞库”这两个名词。
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
