Name: <meta charset="utf8"></head><body>Auteur in de schijnwerpers: Tijd- en kosteneffectief fibrinogeen-PAGE voor fibrinogenolytische studies
Uploaded: 2024-04-19T13:50:00
Description: Read this Scientific Article Protocol about Rapid Separation and Display of Active Fibrinogenolytic Agents in Sipunculus nudus through Fibrinogen-Polyacrylamide Gel Electrophoresis at JoVE.com

<meta charset="utf8"></head><body>Onthulling van fibrinogenolytische middelen in pindawormen met fibrinogeen-PAGE

<ol>
	<li>Bikdeli, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=COVID-19+and+thrombotic+or+thromboembolic+disease:+Implications+for+prevention,+antithrombotic+therapy,+and+follow-up.">COVID-19 and thrombotic or thromboembolic disease: Implications for prevention, antithrombotic therapy, and follow-up.</a> J Am Coll Cardiol. 75 (23), 2950-2973 (2020).</li><li>Tsivgoulis, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thrombolysis+for+acute+ischaemic+stroke:+current+status+and+future+perspectives.">Thrombolysis for acute ischaemic stroke: current status and future perspectives.</a> Lancet Neurol. 22 (5), 418-429 (2023).</li><li>Tang, M., Hu, C., Lin, H., Yan, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrinolytic+drugs+induced+hemorrhage:+mechanisms+and+solutions.">Fibrinolytic drugs induced hemorrhage: mechanisms and solutions.</a> Blood Coagul Fibrinolysis. 34 (5), 263-271 (2023).</li><li>Lu, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification,+characterization,+and+chemical+modification+of+Bacillus+velezensis+SN-14+fibrinolytic+enzyme.">Purification, characterization, and chemical modification of Bacillus velezensis SN-14 fibrinolytic enzyme.</a> Int J Biol Macromol. 177, 601-609 (2021).</li><li>Abu-Tahon, M. A., Abdel-Majeed, A. M., Ghareib, M., Housseiny, M. M., Abdallah, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thrombolytic+and+anticoagulant+efficiencies+of+purified+fibrinolytic+enzyme+produced+from+Cochliobolus+hawaiiensis+under+solid-state+fermentation.">Thrombolytic and anticoagulant efficiencies of purified fibrinolytic enzyme produced from Cochliobolus hawaiiensis under solid-state fermentation.</a> Biotechnol Appl Biochem. 70 (6), 1954-1971 (2023).</li><li>Lin, W., Lin, H., Xin, P., Yan, H., Kang, B., Tang, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Comprehensive+approach+to+analyze+the+cell+components+of+cerebral+blood+clots.">A Comprehensive approach to analyze the cell components of cerebral blood clots.</a> J Vis Exp. (197), e65791 (2023).</li><li>Tang, M., Lin, H., Hu, C., Yan, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Affinity+purification+of+a+fibrinolytic+enzyme+from+Sipunculus+nudus.">Affinity purification of a fibrinolytic enzyme from Sipunculus nudus.</a> J Vis Exp. (196), e65631 (2023).</li><li>. Plasmin affinity purification method based on agarose gel and plasmin and application thereof Available from: <a target="_blank" href="https://patents.google.com/patent/CN116103270A/en?oq=CN202310108470">https://patents.google.com/patent/CN116103270A/en?oq=CN202310108470</a> (2023)</li><li>Walton, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+fibrin+plate+method+for+the+assay+of+plasminogen+activators.">An improved fibrin plate method for the assay of plasminogen activators.</a> Clinica Chimica Acta. 13 (5), 680-684 (1966).</li><li>Ngere, J. B., Ebrahimi, K. H., Williams, R., Pires, E., Walsby-Tickle, J., McCullagh, J. S. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ion-exchange+chromatography+coupled+to+mass+spectrometry+in+life+science,+environmental,+and+medical+research.">Ion-exchange chromatography coupled to mass spectrometry in life science, environmental, and medical research.</a> Anal Chem. 95 (1), 152-166 (2023).</li><li>Kato, S., Takeuchi, K., Iwaki, M., Miyazaki, K., Honda, K., Hayashi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chitin-+and+streptavidin-mediated+affinity+purification+systems:+A+screening+platform+for+enzyme+discovery.">Chitin- and streptavidin-mediated affinity purification systems: A screening platform for enzyme discovery.</a> Angew Chem Int Ed Engl. 62 (31), e202303764 (2023).</li><li>Sharma, N., Sharma, R., Rajput, Y. S., Mann, B., Singh, R., Gandhi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+methods+for+milk+proteins+on+polyacrylamide+gel+electrophoresis:+Critical+analysis+and+options+for+better+resolution.">Separation methods for milk proteins on polyacrylamide gel electrophoresis: Critical analysis and options for better resolution.</a> International Dairy Journal. 114, 104920 (2021).</li><li>. Preparation and application of natural fibrinolytic enzyme from peanut worm Available from: <a target="_blank" href="https://patents.google.com/patent/CN109295042A/en">https://patents.google.com/patent/CN109295042A/en</a> (2019)</li></ol>

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	<tbody>
		<tr>
			<td>1&#160; M Tris-HCl (pH 6.8)</td>
			<td>Solarbio</td>
			<td>T1020</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 M Tris-HCl (pH 8.8)</td>
			<td>Solarbio</td>
			<td>T1010</td>
			<td></td>
		</tr>
		<tr>
			<td>30% Acrylamide/Bis-acrylamide</td>
			<td>Biosharp</td>
			<td>BL513B</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium persulfate</td>
			<td>XiLONG SCIENTIFIC</td>
			<td>7727-54-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Beaker</td>
			<td>PYREX</td>
			<td>2-9425-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge Tube (1.5 mL)</td>
			<td>Biosharp</td>
			<td>BS-15-M</td>
			<td></td>
		</tr>
		<tr>
			<td>Constant Temperature Incubator</td>
			<td>JINGHONG</td>
			<td>JHS-400</td>
			<td></td>
		</tr>
		<tr>
			<td>Coomas Brillant Blue Stainning solution</td>
			<td>Beyotime</td>
			<td>P0017F</td>
			<td></td>
		</tr>
		<tr>
			<td>Electronic Analytical Balance</td>
			<td>DENVER</td>
			<td>TP-213</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibrinogen</td>
			<td>Solarbio</td>
			<td>F8051</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel loading pipette tips, Bulk</td>
			<td>Biosharp</td>
			<td>BS-200-GTB</td>
			<td></td>
		</tr>
		<tr>
			<td>Homogenizer</td>
			<td>AHS</td>
			<td>ATS-1500</td>
			<td></td>
		</tr>
		<tr>
			<td>Horizontal rotation oscillator</td>
			<td>NuoMi</td>
			<td>NMSP-600</td>
			<td></td>
		</tr>
		<tr>
			<td>Milli-Q Reference</td>
			<td>Millipore</td>
			<td>Z00QSV0CN</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini-PROTEAN Tetra Cell</td>
			<td>BIO-RAD</td>
			<td>165-8000~165-8007</td>
			<td></td>
		</tr>
		<tr>
			<td>N,N,N&#39;,N&#39;-Tetramethylethylenediamine</td>
			<td>Sigma</td>
			<td>T9281</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette Tip (1 mL)</td>
			<td>Axygene</td>
			<td>T-1000XT-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette Tip (10 &#181;L)</td>
			<td>Axygene</td>
			<td>T-10XT-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette Tip (200 &#181;L)</td>
			<td>Axygene</td>
			<td>T-200XT-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipettor (1 mL)</td>
			<td>Thermo Fisher Scientific</td>
			<td>ZY18723</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipettor (10&#181;L)</td>
			<td>Thermo Fisher Scientific</td>
			<td>ZX98775</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipettor (200 &#181;L)</td>
			<td>Thermo Fisher Scientific</td>
			<td>ZY20280</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipettor (50 &#181;L)</td>
			<td>Thermo Fisher Scientific</td>
			<td>ZY15331</td>
			<td></td>
		</tr>
		<tr>
			<td>Refrigerated Centrifuge</td>
			<td>cence</td>
			<td>H1650R</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>V900859</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Solarbio</td>
			<td>T8060</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>Solarbio</td>
			<td>T8230</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Solarbio</td>
			<td>T8200</td>
			<td></td>
		</tr>
	</tbody>
</table>

rapid separation and display of active fibrinogenolytic agents in sipunculus nudus through fibrinogen-polyacrylamide gel electrophoresis

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>Auteur in de schijnwerpers: Tijd- en kosteneffectief fibrinogeen-PAGE voor fibrinogenolytische studies

Snelle scheiding en weergave van actieve fibrinogenolytische middelen in Sipunculus nudus door middel van fibrinogeen-polyacrylamide-gelelektroforese

Our research is focused on the fibrinolytic system. In this study, we are trying to simplify the mass 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. Overall, this mass is time and cost effective. With our findings, the discovery of neuro-fibrinogenolytic agents will become simple and can speed up the development of novel 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 fibrinogenolytic agents that can be useful for therapeutics.

Read this Scientific Article Protocol about Rapid Separation and Display of Active Fibrinogenolytic Agents in Sipunculus nudus through Fibrinogen-Polyacrylamide Gel Electrophoresis at JoVE.com

Fibrinogenolytic agents that can dissolve fibrinogen directly have been widely used in anti-coagulation treatment. Generally, identifying new ...

rapid-separation-display-active-fibrinogenolytic-agents-sipunculus

Research

JoVE Journal

Biochemistry

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.

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

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.

Bereiding van pindawormhomogenaat en gel voor fibrinogeen-PAGE om fibrinogenolytische middelen te identificeren

Fibrinogen-PAGE for Fibrinogenolytic Agent Analysis in Peanut Worm Homogenate

To begin, prepare a gel for fibrinogen phage 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 5 microliters of 5x non-denaturing loading buffer with 20 microliters each of fibrinogenolytic 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 kilo-daltons or higher.