Description
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Transfection Reagent for CHO Cells (Chinese hamster ovary cells, CCL-61)
- Two component formulation enhances lipid mediated transfection efficiency
- Optimized easy-to-use transfection protocol provided for transfection of siRNA, DNA, mRNA, and microRNA
- Kit includes Transfection Enhancer reagent and recommended transfection protocol
- High transfection efficacy in the presence of serum
- Expand your RNAi application with a reagent optimized for delivery of both siRNA and plasmid
- Download in vitro CHO transfection protocol: [PDF]
- Download CHO CRISPR/Cas9 transfection protocol: [PDF]
- Download PowerPoint presentation for CHO cells transfection kit: [PPT]
- Developed and manufactured by Altogen Biosystems
Transfection Efficiency:
Reagent exhibits at least 90% transfection efficiency of siRNA delivery. Transfection efficiency was determined by qRT-PCR.
Transfection Protocol and MSDS:
Download Altogen Biosystems Transfection Protocol: [PDF]
Download MSDS: [PDF]
CHO Cell Line:
Production of recombinant protein therapeutics is considered one of the most innovative biotechnological advances and can be highly effective for treatment of many diseases and conditions, including cancers, genetic and autoimmune disorders, and infectious diseases. Various non-human mammalian cell lines are generally used for the production of therapeutic proteins, with Chinese hamster ovary (CHO) cell line being the most important because of its ability to perform human-like protein folding. The Chinese Hamster Ovary (CHO) tissue cell line was first established in 1957 using Chinese Hamster (Cricetulus griseus) ovary cells and is one of the most well-used cell lines for clinical research, and for the synthesis of therapeutic proteins. Also, this mammalian cell line is highly utilized in transfection as well as in large-scale recombinant protein production. CHO cells exhibit an epithelial-like cell morphology and are extensively employed in biomedical research and biotechnology R&D applications. Altogen Biosystems offers the lipid-mediated CHO transfection reagent kit for Chinese hamster ovary cells that yields nearly 90% transfection efficiency.
Data:
Figure 1. GAPD mRNA levels were quantified using real-time RT-PCR in the CHO cells transfected with siRNAs targeting GAPD or non-silencing siRNA. Forty-eight hours post-transfection, the cells were harvested and analyzed by real-time RT-PCR for GAPDH mRNA expression levels. Data were normalized against the 18S rRNA signal. Control samples were either mock-transfected or untreated. Values are normalized to untreated sample. Data are means ± SD (n=3).
Figure 2. Protein expression of GAPDH in CHO cells. DNA plasmid expressing GAPDH or siRNA targeting GAPDH were transfected into CHO cells following Altogen Biosystems transfection protocol. At 72 hours post-transfection the cells were analyzed by Western Blot for protein expression levels (normalized by total protein, 10 µg of total protein loaded per each well). Untreated cells used as a negative control.
Selected in vivo transfection product citations (ALTOGEN® IN VIVO Transfection Kits used in the following publications):
- Nature. 2008 454(7203):523-7. Innate immunity induced by composition-dependent RIG-I …Saito et al [PDF]
- Am J Pathology. 2010 177(4):1870-80. Role of ocular complement factor H in a murine model … Lyzogubov et al [PDF]
- Nature Biotechnology. 2011 29(4):341-5. Delivery of siRNA to the mouse brain by … Alvarez-Erviti et al [PDF]
- Gastroenterology. 2011 141(2) Differential type I interferon-mediated autophagic trafficking … Desai et al [PDF]
- PLoS Pathog. 2014 10(10) Exosomes from hepatitis C infected patients transmit HCV … Bukong et al [PDF]
CHO Transfection Kit
Altogen Biosystems:
Altogen Biosystems transfection and electroporation products for life sciences and cancer research. Transfection reagents are developed for individual cancer cell line and transfection protocols are optimized for maximum delivery efficiency. Advanced formulation of reagents and optimized transfection protocols provide efficient intracellular delivery of proteins, DNA, mRNA, shRNA, siRNA, and other negatively charged biomolecules in vitro and in vivo. Read more about transfection technology at Altogen’s Transfection Resource.
Altogen Labs Research Services:
Altogen Labs provides GLP-compliant contract research studies for pre-clinical research, IND applications, and drug development. Biology CRO services include: Xenograft models (30+), development of stable cell lines, ELISA assay development, cell-based and tissue targeted RNAi studies, safety pharm/tox assays, and other studies (visit AltogenLabs.com).
Volume Options:
- 0.5 ml (Catalog #2055)
- 1.5 ml (Catalog #2066)
- 1.5 ml CRISPR (Catalog #2128)
- 8.0 ml (Catalog #7029)
AltogenBiosystems是一家开发和制造用于生命科学研究,药物发现和开发的转染试剂盒的生物技术公司。转染试剂盒针对特定癌细胞系和原代细胞培养进行了优化,可将生物分子有效递送到靶组织中。通过先进的试剂配方和优化的转染方案实现体外(癌细胞系)和体内(动物组织靶向试剂、癌细胞系)递送货物分子,包括质粒DNA,各种类型的RNA(mRNA,siRNA,shRNA,microRNA),蛋白质和小分子研究。
Altogen生命科学公司致力于研发,生产和销售特定细胞系的转染试剂,用于细胞间生物分子的传递,并通过对转染试剂类型的设计将siRNA和质粒DNA有效地转入不同的细胞系和原代细胞内。Altogen公司开发的聚合物,脂质体,纳米粒子为基础的转染技术分别针对分子生物学,组合化学,和细胞生物学而分别应用。Altogen定制服务提供符合GLP要求定制研究服务,包括代稳定的细胞系,细胞银行和冷冻保存,焦磷酸测序,克隆,RNA干扰(RNAi)和基因沉默服务,发展分析,siRNA文库筛选,并转染服务。稳定的肿瘤细胞株和原代细胞的产生,可以是非常昂贵和费时。该公司的细胞培养科学家的细胞株的选择,无论是利息或shRNA表达载体的稳定表达的基因改造。标准的RNAi技术服务,包括设计与合成的siRNA的利益,验证siRNA的沉默效率,siRNA转染条件的优化,使高效的基因沉默细胞系或原代培养细胞的靶基因。转染培养细胞的瞬时或稳定的引入外源性分子和遗传物质(即RNA或DNA),通常是在生物实验室用来研究基因功能,基因表达的调节,生化映射,突变分析,和蛋白质的生产。科学家利用各种载体分子,这种分子,使质粒DNA(PDNA),信使RNA(mRNA),短干扰RNA(siRNA),小分子RNA(miRNA)的,并进入肿瘤细胞株和原代细胞的蛋白质的基因交付。不幸的是,无单提货的方法或转染试剂,可以适用于所有类型的细胞,细胞的细胞毒性和转染效率显着不同,取决于试剂,协议,并正在利用细胞类型。Altogen生物系统公司提供超过60种类型的细胞的预优化转染试剂盒。纳米粒子,脂质和聚合物基ALTOGEN®在体内转染试剂,使交付功能的RNA和DNA分子在体内。PEG脂质体在体内输送系统减少由于PEG修饰的先天免疫反应,并提供高效的siRNA转染的DNA,并在体内的蛋白质。由科学“杂志(2010年12月17日):PEG脂质体在体内转染试剂盒siRNA的特色Altogen生物系统功能的特定细胞系转染试剂盒
120+细胞转染试剂和活体组织靶向试剂盒制造商AltogenBiosystems是一家生物技术公司,开发和制造用于生命科学研究、药物发现和开发的转染试剂盒。Altogen®体内转染试剂可有效地将生物分子导入靶组织。细胞转染试剂盒针对特定的癌细胞系和原代细胞进行了优化。通过先进的试剂配方和优化的转染方案实现货物分子(DNA、RNA、蛋白质)的高效传递。AltogenBiosystems利用高分子化学、分子和细胞生物学的专业知识,开发了新的体内外给药技术。转染是将外源分子导入培养细胞中,常用于研究基因功能、基因表达调控、生化定位和蛋白质生产。不幸的是,由于细胞毒性和转染效率的差异很大,并且取决于所使用的试剂、方案和细胞类型,因此没有一种单一的传递方法或转染试剂可应用于所有类型的细胞。AltogenBiosystems为120多个癌细胞系和原代细胞类型提供优化的转染试剂盒和电穿孔产品。体内转染试剂可实现组织靶向给药。Altogen的转染试剂盒包括用于体外(癌细胞系)和体内(用于动物研究的组织靶向试剂)转染的转染增强剂试剂和转染复合物冷凝器。Altogen实验室提供符合GLP的实验室合同研究服务。我们的生物CRO服务包括异种移植物的疗效、IND应用的pharm/tox研究和安全性测试、分析开发(ELISA、IC-50、qPCR)、90多个异种移植物动物模型、RNAi和基因沉默服务。Altogen的细胞培养科学家通过在28天内培育出稳定的细胞系,将选择的细胞系转化为稳定表达感兴趣的基因。
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不知单纯使用冰浴中超声裂解够了吗?还是说要选用裂解液呢?最主要的就是我不知道该怎么赔这个裂解液,使其COX活性不产生影响
样品处理:
a. 植物组织:取新鲜或-70℃冻存100mg组织在液氮中研磨,把粉末加入到1ml裂解液中混匀。
b. 动物组织:取新鲜或-70℃冻存100mg组织加1ml裂解液,用组织研磨杵或匀浆器匀浆处理。
c. 贴壁细胞:直接在培养板中加入裂解液裂解细胞,每106细胞加1ml 裂解液。用取样器吹打混匀。
d. 细胞悬液:离心收集细胞。每106动物、植物和酵母细胞或每107细菌细胞加1ml裂解液混匀。
e. 血液处理:取0.2-1ml新鲜血液加3倍体积红细胞裂解液,混匀后室温放置10分钟,10000rpm离心1分钟。弃上清,若沉淀含有红细胞,可加入2倍体积红细胞裂解液重复裂解步骤。离心后沉淀加入1 ml裂解液混匀。
将处理后的样品在室温放置5分钟,使得核酸蛋白复合物完全分离。
向匀浆样品中加0.2ml氯仿,盖好管盖,剧烈振荡15秒,室温放置3-5分钟。
2-8℃ 12000 rpm离心10分钟。RNA主要在上层无色的水相中,把水相转移到新管中,不要吸到沉淀。
吸附柱前处理:在吸附柱中加入500ul 洗柱液,室温放置2分钟,2-8℃ 12,000 rpm离心2min,弃废液。6. 第4步收集的上清中加入200ul无水乙醇混匀,加入吸附柱静置2分钟,2-8℃ 12000rpm离心2min,弃废液。7. 向吸附柱中加入600ul漂洗液(使用前请先检查是否已加入无水乙醇),2-8℃ 12,000 rpm离心2min,弃废液。8. 向吸附柱中加入600ul漂洗液,2-8℃ 12,000 rpm离心2min,弃废液。9. 12000rpm离心2min,弃掉收集管,将吸附柱置于室温放置数分钟将吸附柱中残余的漂洗液去除。10. 将吸附柱放入新管中,向膜中央滴加50-100ul RNase free ddH2O,室温放置5min,12000rpm室温离心2min即得到RNA。
注意事项:
所有相关器皿耗材都应为RNase-free产品,操作过程要小心,带口罩、手套避免环境中RNA酶污染样品。
RNA在水溶液中OD值可能在1.5-1.9之间,但这并不表示RNA不纯,需电泳检测。
A.优点:
多重分析——一次分析可以检测16种干细胞相关的TF
定量比较——二个样本的差异可以定量分析和比较
步骤简单——探针温育、柱分离、板杂交和HRP检测
无需贵重仪器——无需如Luminex那样的贵重仪器
B.原理:
干细胞转录因子活性多重检测阵列试剂用于同时检测多种TF活性。该技术中,基于TFDNA结合位点的一致性序列,制备一系列生物素标记的探针。当探针混合物与核提取物一起温育时,每个探针寻找相应的TF,形成TF/探针复合物,通过柱离心纯化可以很容易与游离探针分开。结合的探针从混合物中分离出来,通过板杂交分析。板孔中预包被上与探针互补的特异序列,捕获的DNA探针进一步用链酶亲和素-HRP检测,化学发光检测仪测定发光强度(RLUs)。
【2】加入裂解液后可以加用超声破碎细胞。
【3】提完细胞的器具可以在显微镜下看一下,剩余的细胞还多不多。如果还多,说明细胞提取的不彻底,还要改进方法,争取提取的细胞彻底一些。
(仅供参考)
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