Puromycin Dihydrochloride (嘌呤霉素)
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产品编号 ： ST551-10mg
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¥ 208.00 元
产品包装:
10mg/ml×1ml 10mg/ml×5ml 250mg
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      Puromycin是来源于Streptomyces alboniger的一种氨基核苷类抗生素，中文名为嘌呤霉素，常用于筛选通过质粒转染/转
化、病毒感染等方法能表达pac基因(puror)的真核或原核多克隆或单克隆细胞。Puromycin不仅用于稳定细胞株的筛选，也用于稳定细胞株的维持。本产品的10mg/ml包装经过滤除菌，可以直接用于细胞培养。
      Puromycin的特点是快速作用于细胞，一般2天内可以杀死99%的不表达pac基因的细胞。
      在革兰氏阳性菌、动物或昆虫细胞中，嘌呤霉素通过抑制蛋白质合成而抑制或杀死细胞。其作用机制为嘌呤霉素是氨酰-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。嘌呤霉素与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。
      Pac基因表达嘌呤霉素N-乙酰转移酶(Puromycin N-acetyl-tranferase)，该基因是在Streptomyces alboniger中发现的。如果表达基因，就会对嘌呤霉素产生抗性，这一特性目前普遍应用于筛选表达pac基因的哺乳动物稳定细胞株等。例如，很多商业化的慢病毒载体都携带pac基因(一般在质粒图谱上标记为puror)，从而利用嘌呤霉素筛选特定基因的稳定表达细胞株。嘌呤霉素也可以用来筛选表达pac基因的大肠杆菌菌株、酵母菌株等。
      化学性质

化学名 (2S)-2-amino-N-[(2S,3S,4R,5R)-5-[6-(dimethylamino)purin-9-yl]-4-hydroxy-2-(hydroxymethyl)oxolan-3-yl]-3-(4-methoxyphenyl)propanamide;dihydrochloride
化学式 C22H29N7O5•2HCl
分子量 544.43
CAS号 58-58-2
纯度 >98%

      本产品10mg/ml包装配制在20mM HEPES(pH7.4)溶液中，浓度为10mg/ml，经0.22μm滤膜过滤除菌，可以直接用于细胞培养。本产品250mg包装为粉末装。
包装清单：

产品编号 产品名称 包装
ST551-10mg Puromycin Dihydrochloride (嘌呤霉素) 10mg/ml×1ml
ST551-50mg Puromycin Dihydrochloride (嘌呤霉素) 10mg/ml×5ml
ST551-250mg Puromycin Dihydrochloride (嘌呤霉素) 250mg
— 说明书 1份

保存条件：
        -20℃保存，至少一年有效。10mg/ml包装避免反复冻融。
注意事项：
        本产品对人体有害，操作时请小心，并注意有效防护以避免直接接触人体或吸入体内。
       本产品仅限于专业人员的科学研究用，不得用于临床诊断或治疗，不得用于食品或药品，不得存放于普通住宅内。
       为了您的安全和健康，请穿实验服并戴一次性手套操作。

1. 推荐工作浓度：推荐的作用于哺乳动物细胞的嘌呤霉素浓度一般为1-10μg/ml，但最佳工作浓度需要通过剂量反应曲线来确定。
表1. 部分常见哺乳动物细胞的推荐工作浓度表

细胞名称 细胞类型 嘌呤霉素浓度
A549 Lung cancer 1-2μg/ml
B16 Mouse melanocytes 1-2μg/ml
ES cell Human embryonic stem cells 0.5-5μg/ml
H1299 Non-small cell lung carcinoma 1-3μg/ml
HEK293 Human embryonic kidney 0.5-3μg/ml
HeLa Human cervical cancer 1-2μg/ml
HepG2 Human hepatocellular carcinoma 0.5-2μg/ml
HT1080 Human fibrosarcoma 0.5-2μg/ml
MCF-7 Human breast cancer 0.5-2μg/ml
MDA-MB-231 Human breast cancer 0.5-5μg/ml
MEF Mouse fibroblasts 1-5μg/ml

2. 嘌呤霉素剂量反应曲线的确定(以shRNA转染或者慢病毒感染为例）
嘌呤霉素的有效筛选浓度跟细胞类型、生长状态、细胞密度、细胞代谢及细胞所处细胞周期等因素相关。为了筛选到稳定表达的shRNA或感染病毒的细胞株，确定杀死未转染/感染细胞的最低浓度嘌呤霉素非常重要。对于初次使用的细胞，一般需要通过实验来确定适合自身实验体系的剂量反应曲线(dose-response curve or kill curve)。
a. 第一天：24孔板中以5~8×104cells/孔的密度接种细胞，接种够量的孔以便进行后续的剂量梯度实验。细胞培养箱内培养过夜。
b. 第二天：在培养过夜后的细胞中更换新鲜配制的筛选培养基，该筛选培养基为含不同浓度嘌呤霉素的新鲜培养基(如0、1、2.5、5、7.5、10μg/ml等)，更换培养基后在细胞培养箱中继续培养。
c. 第三天后：由于嘌呤霉素可以快速作用于细胞，一般2天内可以杀死99%的未表达pac基因的细胞，所以在加嘌呤霉素后的1-2天就可以进行观察细胞存活率，从而确定有效杀死正常细胞的药物最低浓度。如果细胞耐药性比较强，需要每日观察，一般4-10天内即可确定嘌呤霉素的最低浓度。
3. 哺乳动物稳定表达细胞株的筛选
转染含有pac基因的质粒或者感染含有该基因的病毒后，即可筛选稳定表达株。
a. 细胞转染或感染48小时后，将细胞置于含有适当浓度嘌呤霉素的新鲜培养基中培养，此为处理组。
注意：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，所以细胞的密度最好不超过25%。建议同时做一个正常细胞的对照组。转染或感染48小时后，如果细胞过密也可以消化后重新接种细胞，培养过夜后即可进行嘌呤霉素筛选。
b. 每隔2-3天，更换含有嘌呤霉素的培养基。
c. 筛选7天后，对照组正常细胞应该100%死亡，处理组中存活的细胞为表达pac基因的细胞。然后根据实验目的进行多克隆或单克隆细胞的筛选。
注意：每日进行细胞生长状态的观察。嘌呤霉素的筛选至少需要24小时，有效浓度嘌呤霉素的筛选周期一般在2-10天。
d. 待细胞可以稳定生长后，嘌呤霉素的浓度可以减半用于后续的培养。在得到稳定表达细胞株后，一般建议嘌呤霉素也须持续加入，并2-3天更换含有嘌呤霉素的新鲜培养液。

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C0351-1ml
10mg/ml×1ml
Polybrene (Hexadimethrine Bromide)
ST551-50mg
10mg/ml×5ml
Puromycin Dihydrochloride (嘌呤霉素)
ST018-1ml
10mg/ml×1ml
Blasticidin S HCl (灭瘟素S)
ST551-250mg
250mg
Puromycin Dihydrochloride (嘌呤霉素)
C0351-50mg
50mg
Polybrene (Hexadimethrine Bromide)
ST081-1ml
50mg/ml×1ml
G418 (遗传霉素)
S0033S
>100次
活性氧检测试剂盒
C0533-0.5ml
0.5ml
Lipo8000™转染试剂
ST008
5ml
Ampicillin (100mg/ml,1000X)
ST1389-50mg
50mg
Hygromycin B(潮霉素B)
P0013B
100ml
RIPA裂解液(强)
C0222
100ml
青霉素-链霉素溶液(100X)