preparing peanut worm homogenate and gel for fibrinogen-page to identify fibrinogenolytic agents

<meta charset="utf8"></head><body>使用 Fibrinogen-PAGE 揭示花生蠕虫中的纤维蛋白原溶解剂

In recent years, due to the continued rise of thrombotic diseases, thrombotic diseases have become a new major global health problem1. At present, antithrombotic drugs are classified into three groups: anti-platelet aggregation drugs, anticoagulants, and thrombolytic drugs. Among them, thrombolytic drugs, such as urokinase (UK), tissue plasminogen activator (tPA), etc., are the only clinically used drugs that can hydrolyze thrombus2. Meanwhile, more safe and effective thrombolytic drugs are being developed by the identification of novel thrombolytic agents3.
However, the identification of novel thrombolytic agents is time-consuming and labor-intensive, which mainly involves the separation/purification and characterization/checking stages. The former is to separate each component, and the latter is to display their fibrino(geno)lytic activities4,5. In previous studies, despite the fact we have successfully isolated a novel fibrino(geno)lytic enzyme (sFE) from the peanut worm (Sipunculus nudus) by affinity chromatography and fibrin(ogen) plate assay6,7,8, these processes are such time and labor-consuming. First, it is necessary to determine whether the peanut worm homogenates have fibrino(geno)lytic activity by the fibrin(ogen) plate method and reaction products based on PAGE9. Then, a series of chromatography, such as ion exchange chromatography, gel filtration chromatography, affinity chromatography, and other methods, have to be carried out for separation and purification10,11. Subsequently, the fibrin plate assay is performed again to check the fibrinogenolytic activity of each separated component. Finally, native-PAGE and sodium dodecyl sulfate (SDS)-PAGE are performed to determine the molecular weight of the active fibrinogenolytic agents12. Therefore, it is critical to rapidly separate and display the active fibrinogenolytic agents.
To rapidly separate and display the active fibrinogenolytic agents in the peanut worm homogenates, a new fibrinogen-PAGE method was developed by combining the PAGE and fibrinogen plate methods, i.e., the substrates of fibrinogenolytic agents fibrinogen were added to the native-PAGE gel. After native-PAGE, the components were separated by their molecular weight. Meanwhile, each active fibrinogenolytic component can be displayed by staining. By this method, we successfully detected more than one active fibrinogenolytic agent of peanut worm&#160;homogenate within 6 h. Moreover, this fibrinogen-PAGE method is time and cost-friendly. Furthermore, this method could be used to study the fibrinogenolytic agents of the other organisms.

<meta charset="utf8"></head><body>作者聚焦：用于纤维蛋白原溶解研究的时间和成本效益的纤维蛋白原-PAGE

通过纤维蛋白原-聚丙烯酰胺凝胶电泳快速分离和展示 Sipunculus nudus 中的活性纤维蛋白原溶解剂

制备花生蠕虫匀浆和凝胶用于纤维蛋白原-PAGE 以鉴定纤维蛋白原溶解剂

preparing-peanut-worm-homogenate-gel-for-fibrinogen-page-to-identify

Research

JoVE Journal

Biochemistry

Preparing Peanut Worm Homogenate and Gel for Fibrinogen-PAGE to Identify Fibrinogenolytic Agents

145 次观看. 首先，称取 50 克花生蠕虫的重量，将它们与 150 毫升盐水一起加入均质器中。以 24， 000 RPM 的转速均质化 60 秒。离心匀浆并收集上清液用于进一步处理。对于纤维蛋白原聚丙烯酰胺凝胶制备，将 0.01 克纤维蛋白原和其他试剂加入 50 毫升玻璃烧杯中。将烧杯在 37 摄氏度下加热 30 分钟。加入丙烯酰胺后，将分离凝胶混合物倒入 10 孔凝胶模具中。

视频: 制备花生蠕虫匀浆和凝胶用于纤维蛋白原-PAGE 以鉴定纤维蛋白原溶解剂

Rapid Separation and Display of Active Fibrinogenolytic Agents in Sipunculus nudus through Fibrinogen-Polyacrylamide Gel Electrophoresis

Fibrinogenolytic agents that can dissolve fibrinogen directly have been widely used in anti-coagulation treatment. Generally, identifying new fibrinogenolytic agents requires the separation of each component first and then checking their fibrinogenolytic activities. Currently, polyacrylamide gel electrophoresis (PAGE) and chromatography are mostly used in the separating stage. Meanwhile, the fibrinogen plate assay and reaction products based PAGE are usually adopted to display their fibrinogenolytic activities. However, because of the spatiotemporal separation of those two stages, it is impossible to separate and display the active fibrinogenolytic agents with the same gel. To simplify the separating and displaying processes of fibrinogenolytic agent identification, we constructed a new fibrinogen-PAGE method to rapidly separate and display the fibrinogenolytic agents of peanut worms (Sipunculus nudus)&#160;in this study. This method includes fibrinogen-PAGE preparation, electrophoresis, renaturation, incubation, staining, and decolorization. The fibrinogenolytic activity and molecular weight of the protein can be detected simultaneously. According to this method, we successfully detected more than one active fibrinogenolytic agent of peanut wormhomogenate within 6 h. Moreover, this fibrinogen-PAGE method is time and cost-friendly. Furthermore, this method could be used to study the fibrinogenolytic agents of the other organisms.

Here, we present a fibrinogen-polyacrylamide gel electrophoresis (PAGE) protocol to rapidly separate and display the fibrinogenolytic agents of Sipunculus nudus.