Holm Tina, Johansson Henrik, Lundberg Pontus, Pooga Margus, Lindgren Maria, Langel Ulo
Department of Neurochemistry, Stockholm University, S. Arrheniusv. 21A, SE-106 91, Stockholm, Sweden.
Nat Protoc. 2006;1(2):1001-5. doi: 10.1038/nprot.2006.174.
More than a decade ago, it was discovered that cationic peptides could traverse the cellular plasma membrane without specific transporter proteins or membrane damage. Subsequently, it was found that these peptides, known as cell-penetrating peptides (CPPs), were also capable of delivering cargos into cells, hence the great potential of these vectors was acknowledged. Today, many different research groups are working with CPPs, which necessitates efforts to develop unified assays enabling the comparison of data. Here we contribute three protocols for evaluation of CPPs which, if used in conjunction, provide complementary data about the amount and mechanism of uptake (fluorometric analysis and confocal microscopy, respectively), as well as the extent of degradation (HPLC analysis of cell lysates). All three protocols are based on the use of fluorescently labeled peptides and can be performed on the same workday.
十多年前，人们发现阳离子肽可以在没有特定转运蛋白或膜损伤的情况下穿过细胞质膜。随后，人们发现这些被称为细胞穿透肽（CPPs）的肽也能够将货物递送至细胞内，因此这些载体具有巨大的潜力得到了认可。如今，许多不同的研究小组都在研究CPPs，这就需要努力开发统一的检测方法，以便对数据进行比较。在此，我们提供了三种评估CPPs的方案，如果结合使用，这些方案可以提供关于摄取量和摄取机制（分别为荧光分析和共聚焦显微镜）以及降解程度（细胞裂解物的HPLC分析）的补充数据。所有这三种方案均基于使用荧光标记的肽，并且可以在同一个工作日内完成。
