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

<meta charset="utf8"></head><body>Presentación de agentes fibrinogenolíticos en gusanos de maní con fibrinógeno-PAGE

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>Autor destacado: Fibrinógeno-PAGE rentable y en tiempo para estudios fibrinogenolíticos

Separación rápida y visualización de agentes fibrinogenolíticos activos en Sipunculus nudus mediante electroforesis en gel de fibrinógeno-poliacrilamida

Preparación de homogeneizado y gel de gusano de maní para fibrinógeno-PAGE para identificar agentes fibrinogenolíticos

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 vistas. Para empezar, pesa 50 gramos de gusanos de cacahuete y añádelos al homogeneizador, junto con 150 mililitros de solución salina. Homogeneizar a 24.000 RPM durante 60 segundos. Centrifugar el homogeneizado y recoger el sobrenadante para procesos posteriores.Para la preparación del gel de poliacrilamida con fibrinógeno, añada 0,01 gramos de fibrinógeno y otros reactivos en un vaso de precipitados de vidrio de 50 mililitros. Caliente el vaso a 37 gr...

Video: Preparación de homogeneizado y gel de gusano de maní para fibrinógeno-PAGE para identificar agentes fibrinogenolíticos

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.