Ubiquitinylation Kit

Enzo Life Sciences 公司所出品的「Ubiquitinylation Kit」內含 Ubiquitin (biotinylated)、Ubiquitin activating enzyme (E1) (His₆-tagged)、以及 Ubiquitin conjugating enzyme (E2) (His₆-tagged) 等試劑，可作用產生 E2-ubiquitin thioester，應用於下列分析實驗中：

• 製造 Ubiquitinylated Protein：搭配適當的 Ubiquitin ligating enzyme (E3) 使用，可應用於目標蛋白的泛素化反應 (ubiquitinylation)。E3 酵素來源可為重組蛋白、純化產物或 Cell lysates。
  

• 測試參與目標蛋白泛素化反應的最佳 E2 酵素： 由於本產品提供多達 11 種不同的 Ubiquitin conjugating enzyme (E2) 試劑，因此可應用於測試參與目標蛋白泛素化反應的最佳 E2 酵素為何。
  

• E3 酵素活性分析：可建立 in vitro 分析平台找尋可作用於特定目標蛋白的 E3 酵素分子。

Ubiquitin cascade showing activation, conjugation, ligation, deconjugation, and recycling steps. Protein ubiquitinylation is achieved through a minimum of three enzymatic steps. In the first step, in an ATP-dependent process, a ubiquitin-activating enzyme (E1) catalyzes the formation of a reactive thioester bond with ubiquitin. This is followed by its subsequent transfer to the active site cysteine of a ubiquitin carrier protein (E2). The specificity of ubiquitin ligation arises from the subsequent association of the E2-ubiquitin thioester with a substrate-specific ubiquitinprotein isopeptide ligase (E3), which facilitates the formation of the isopeptide linkage between ubiquitin and its target protein. Western Blot of Thioester Assays (TE +ve/-ve controls) for all E2 conjugating enzymes provided. Biotinylated-ubiquitin-enzyme conjugates were detected by Western Blotting using Streptavidin-HRP detection system as described in "Manual". A: UbcH1, B: UbcH2, C: UbcH3, D: UbcH5a, E: UbcH5b, F: UbcH5c, G: UbcH6, H: UbcH7, I: UbcH8, J: UbcH10, K: Ubc13/MMS2, M: Biotinylated SDS markers (Sigma, SDS-6B). Results demonstrate the formation of ubiquitin thioester linked E2 conjugates of the expected size in all TE +ve control reactions. The absence of such conjugates in TE -ve control reactions demonstrates that their formation is ATP dependent (required for E1 activation).

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