耗材描述
Acrylodan
产品活性:Acrylodan 可与硫醇反应,对 Cys34 周围的结合口袋内的局部环境双极性和动力学敏感。
产品来源: https://www.medchemexpress.cn/acrylodan.html
研究领域:Others
作用靶点:Fluorescent Dye
In Vitro: Guidelines (Following is our recommended protocol. This protocol only provides a guideline, and should be modified according to your specific needs).
Purification and Labeling of Act1:
1. 20-fold molar excess of acrylodan (Anaspec) was added to the polymerized actin and incubated on ice overnight.
2. The labeled actin is centrifuged (2 h, 4 °C, 100,000 × g) then the pellet was resuspended in 10 mm Tris, pH 7.5, 0.5 mm β-mercaptoethanol, 0.2 mm CaCl2, 50 μm ATP and dialyzed against the same buffer for 70 h at 4 °C.
3. The supernatant was collected and the actin concentration and degree of labeling were calculated using the extinction coefficient 26,600 M 1 cm 1 for actin at 290 nm, 18,500 M 1 cm 1 or acrylodan at 385 nm, and 71,000 M 1 cm 1 for Alexa Fluor 488 at 494 nm.
4. Acrylodan-labeled Act1 (AcrylAct1NAT) is dramatically different than that of EDTA-unfolded AcrylAct1 (AcrylAct1I3), which has a very similar emission spectrum to CCT-bound AcrylAct1.
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