手机验证
大量现货
Puromycin(Solution 10 mg/mL)
58-58-2
有效期1年
翌圣生物科技(上海)股份有限公司
-20度
1×1 mL
产品信息
产品名称 |
产品编号 |
规格 |
价格 |
促销价 |
Puromycin (Solution 10 mg/mL) 嘌呤霉素盐酸盐溶液 |
60209ES10 |
1×1 mL |
495 |
/ |
60209ES50 |
5×1 mL |
1,495 |
/ |
|
60209ES60 |
10×1 mL |
2,385 |
1,588 |
|
60209ES76 |
50×1 mL |
7,985 |
5288 |
产品描述
嘌呤霉素(Puromycin)是由白黑链霉菌(Streptomyces alboniger)发酵代谢产生的一种氨基糖苷类抗生素,通过抑制蛋白质合成而杀死革兰氏阳性菌,各种动物和昆虫细胞。某种特殊情况下有效作用大肠杆菌。作用机制在于嘌呤霉素是氨酰-tRNA分子3´末端的类似物,能够与核糖体的A位点结合并掺入到延伸的肽链中。嘌呤霉素同A位点结合后,不会参与随后的任何反应,从而导致蛋白质合成的提前终止并释放出C-末端含有嘌呤霉素的不成熟多肽。
嘌呤霉素产生菌Streptomyces alboniger内发现的pac基因编码嘌呤霉素N-乙酰转移酶(PAC),赋予机体对嘌呤霉素产生抗性。这一特性如今普遍应用于筛选特定携带pac基因质粒的哺乳动物稳定转染细胞株。
嘌呤霉素在细胞稳转株筛选中的普遍应用与慢病毒载体的特性有关,现在商业化的慢病毒载体多数都携带pac基因。在某些特定情况下,嘌呤霉素亦可以用来筛选转化携带pac基因质粒的大肠杆菌菌株。
本品是无菌的、溶于蒸馏水的嘌呤霉素盐酸盐溶液,浓度为10 mg/mL(10 mg/mL in H2O),可直接用培养基或其他缓冲溶液稀释使用,适用于细胞培养,常用工作浓度为1~10 µg/mL。
产品性质
CAS号(CAS NO.) | 58-58-2 |
分子式(Formula) | C22H29N7O5·2HCl |
分子量(Molecular weight) | 544.43 g/mol |
外观(Appearance) | 溶液 |
浓度 (Concentration) | 10 mg/mL(溶于水) |
纯度(Purity) | ≥95% |
结构式(Structure) |
细胞系 | 嘌呤霉素浓度 | 参考文献 |
B16(小鼠黑素细胞) | 1~2 μg/mL | 【1】,【2】 |
HEK293(人胚胎肾细胞) | 0.5~10 μg/mL | 【3】 |
HeLa(人宫颈癌细胞) | 1~10 μg/mL | 【4】,【5】 |
MEF(小鼠成纤维细胞) | 1-5 μg/mL | 【4】 |
HepG2(人肝细胞癌) | 0.5~5 μg/mL | 【6】,【7】 |
A549(肺癌细胞) | 1.5 μg/mL | 【8】 |
人胚胎干 (ES) 细胞 | 0.5~5 μg/mL | 【9】 |
2. 嘌呤霉素杀灭曲线的确定(以shRNA转染或者慢病毒转导为例)
嘌呤霉素有效筛选浓度跟细胞类型、生长状态、细胞密度、细胞代谢情况及细胞所处细胞周期位置等有关。为了筛选到稳定表达的shRNA细胞株,确定杀死未转染/转导细胞的最低浓度嘌呤霉素至关重要。建议初次做实验的客户一定要建立适合自身实验体系的杀死曲线(kill curve)。
1)Day 1:24孔板内以5~8×104 cells/孔的密度铺板,铺足够量的孔,以确保后续的梯度实验的进行,37℃细胞孵育过夜。
2)Day 2:①准备筛选培养基:含不同浓度嘌呤霉素的新鲜培养基(如0~15 μg/mL,至少5个梯度);②往孵育过夜后的细胞内更换新鲜配制的筛选培养基;之后37℃孵育细胞。
3)Day 4:更换新鲜的筛选培养基,并观察细胞存活率。
4)根据细胞的生长状态,约2-3天更换新鲜的筛选培养基。
5)每日监测细胞,观察存活细胞率,确定抗生素筛选开始4~6天内有效杀死非转染或所有非转导细胞的药物最低浓度。
3. 哺乳动物稳定转染细胞株的筛选
等转染含有pac基因的质粒后,细胞在含有嘌呤霉素的培养基中增殖,以筛选出稳定转染子。
1)细胞转染48 h后,将细胞(原样或稀释)置于含有适当浓度嘌呤霉素的新鲜培养基中培养。
【注】:当细胞处于分裂活跃期时,抗生素作用最明显。细胞过于密集,抗生素产生的效力会明显下降。最好进行细胞分盘使其密度不超过25%。
2)每隔2~3天,移除和更换含有嘌呤霉素的培养基。
3)筛选7天后评估细胞形成的病灶。病灶可能需要额外的一周或者更多时间,这依赖于宿主细胞系和转染筛选效率。
【注】:每日进行细胞生长状态的观察。嘌呤霉素的筛选至少需要48 h,有效浓度嘌呤霉素的筛选周期一般在3-10天。
4)转移和放置5~10个抗性克隆到一个35 mm的培养皿中,用选择培养基继续培养7天。此次富集培养是为日后的细胞毒性实验做准备。
注意事项
1. 嘌呤霉素为有毒化合物,操作时请小心拿放。
2. 为了您的安全和健康,请穿实验服并戴一次性手套操作。
3. 本产品仅作科研用途!
参考文献
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【9】Paatero A O , Hilkka T , Happonen L J , et al. Bacteriophage Mu integration in yeast and mammalian genomes[J]. Nucleic Acids Research, 2008, 36(22):e148-e148.
客户使用本产品发表的科研文献(部分)
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