Suc-Leu-Leu-Val-Tyr-AMC (solution) 

Suc-Leu-Leu-Val-Tyr-AMC (solution) is a membrane-permeable calpain-specific fluorogenic substrate (Ex/Em = 390/480 nm) ^[1].
Solvent and concentration: DMSO: 20 mM

1. 溶液配制^[1]
1.1 储备液
储存：分装后，置于 -20°C 或 -80°C 避光保存。避免反复冻融。
1.2 工作液配制
用检测缓冲液稀释 (根据实验优化)。可根据实际情况稀释相应的储备液。注意，如果溶剂为 DMSO，则必须考虑 DMSO 的细胞毒性，并应制备溶剂对照；如果溶剂为纯水，则工作液在加入细胞前需要过滤并除菌。
注意：工作液应立即配制并使用，并避光保存。

2.细胞染色
2.1 将 L929 细胞以 10⁵ 个细胞/孔 200 μL 的密度接种于 96 孔板中。
2.2 向所有孔中加入 200 μL 检测缓冲液 (115 mM NaCl、1 mM KH₂PO₄、5 mM KCl、2 mM CaCl₂、1.2 mM MgSO₄、25 mM HEPES 钠缓冲液 (pH 7.4) ) =。
2.3 向细胞中加入 Suc-Leu-Leu-Val-Tyr-AMC (62.5 μM)。
2.4 将培养板置于荧光计中，37°C 孵育，加入底物后立即开始每隔 5 分钟测量一次荧光。

Suc-Leu-Leu-Val-Tyr-AMC (Suc-LLVY) 是一种膜渗透性钙蛋白酶特异性荧光底物，肽基-7-氨基键的蛋白水解释放出高荧光的 7-氨基-4-甲基香豆素 (AMC) 部分^[1]。评估了 TGF-β 对这些底物 (例如 Suc-Leu-Leu-Val-Tyr-AMC) 水解的影响。胆道上皮 H69 细胞与 10、1、0.1 或 0 ng/mL TGF-β 孵育 24 小时。然后在细胞溶质提取物中用荧光法评估底物水解。suc-LLVY-AMC、z-LLE-AMC 和 z-LLL-AMC 水解的基础活性分别为 1.12、8.33 和 14.52 nmol AMC/mg protein/min^[2]。

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

763.89

C₄₀H₅₃N₅O₁₀

94367-21-2

Room temperature in continental US; may vary elsewhere.

Please store the product under the recommended conditions in the Certificate of Analysis.

• [1]. Roberta L. Debiasi, et al. Reovirus-Induced Apoptosis Is Preceded by Increased Cellular Calpain Activity and Is Blocked by Calpain Inhibitors. J Virol. 1999 Jan; 73(1): 695-701.  [Content Brief]

  [2]. Tadlock L, et al. Transforming growth factor-beta inhibition of proteasomal activity: a potential mechanism of growth arrest. Am J Physiol Cell Physiol. 2003 Aug;285(2):C277-85. Epub 2003 Mar 19.  [Content Brief]

  [3]. Gardner RC, et al. Characterization of peptidyl boronic acid inhibitors of mammalian 20 S and 26 S proteasomes and their inhibition of proteasomes in cultured cells. Biochem J. 2000 Mar, 2:447-54.  [Content Brief]