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

Our research is focused on the fibrinolytic system. In this study, we are trying to simplify the method for screening of fibrinogenolytical agents in peanut worms using fibrinogen-PAGE technique. We have developed a new fibrinogen-PAGE method by combining the PAGE and fibrinogen plate methods.

Compared to the other technics currently used, our protocol consists of a 10 year study separate and display the active fibrinogenolytic genes within the same pathogen. Moreover, this method is time and cost-friendly. With our findings, the discovery of neuro-fibrinogenolytic agents will become simple and can speed up the development of novel thrombolytic drugs that can be used to treat a variety of cardiovascular and other diseases.

Our laboratory will focus on studying the coagulation and the fibrinogenolytic systems in different organisms to isolate and screen for novel thrombolytic agents that can be useful for therapeutics. To begin, weigh 50 grams of peanut worms and add them into the homogenizer, along with 150 milliliters of saline. Homogenize at 24, 000 RPM for 60 seconds.

Centrifuge the homogenate and collect the supernatant for further processes. For fibrinogen polyacrylamide gel preparation, add 0.01 grams of fibrinogen and other reagents into a 50 milliliter glass beaker. Warm the beaker at 37 degrees Celsius for 30 minutes.

After adding acrylamide, pour the separating gel mixture into a 10 well gel mold. Then add two milliliters of double-distilled water to the gel mold and incubate it for 30 minutes. Invert the mold to remove the water.

Next, prepare the loading gel similarly and pour it onto the same mold as earlier. Finally, insert the comb for well formation. To begin, prepare a gel for fibrinogen-PAGE and place it in the electrophoresis tank together with the glue mold.

Pour 1, 300 milliliters of electrophoresis solution into the tank and take out the comb from the gel. Mix five microliters of 5X non-denaturing loading buffer with 20 microliters each of fibrinolytic enzyme and peanut worm homogenate. Load the mixture into the prepared gel, and initiate the electrophoresis at 80 volts for 30 minutes or 120 volts for 20 minutes.

Remove the gel from the glue mold and transfer it into a plastic box. Add Tris-HCL and Triton into the box and incubate it on a shaker at 60 to 70 RPM for 10 minutes. Remove the wash buffer and incubate the gel with 50 milliliters of 0.05 molar Tris-HCL.

After removing the buffer, incubate the gel with 50 milliliters of staining, followed by de-colorization solution at 60 to 70 RPM for 30 minutes each. The peanut worm homogenate displayed bands corresponding to molecular weights of 25, 33, 43, and 70 kilodaltons or higher.