<meta charset="utf8"></head><body>Enthüllung von fibrinogenolytischen Wirkstoffen bei Erdnusswürmern mit Fibrinogen-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>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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>Autor im Rampenlicht: Zeit- und kosteneffiziente Fibrinogen-PAGE für fibrinogenolytische Studien

Schnelle Trennung und Darstellung von aktiven fibrinogenolytischen Wirkstoffen in Sipunculus nudus durch Fibrinogen-Polyacrylamid-Gelelektrophorese

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

249 Aufrufe.  Quanzhou First Hospital Affiliated to Fujian Medical University. Unsere Forschung konzentriert sich auf das fibrinolytische System. In dieser Studie versuchen wir, die Masse für das Screening von fibrinogenolytischen Wirkstoffen in Erdnusswürmern mit Hilfe der Fibrinogen-PAGE-Technik zu vereinfachen. Wir haben eine neue Fibrinogen-PAGE-Methode entwickelt, indem wir die PAGE- und die Fibrinogenplatten-Methode kombinieren.Im Vergleich zu den anderen derzeit verwendeten Techniken besteht unser Protokoll aus einer 10-...

Serie: Autor im Rampenlicht: Zeit- und kosteneffiziente Fibrinogen-PAGE für fibrinogenolytische Studien

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.