This research was funded by the Science and Technology Bureau of Xiamen City (No. 3502Z20227197), the Science and Technology Bureau of Fujian Province (No. 2019J01070; No. 2022J01311), and High-level Talents Innovation and Entrepreneurship Project of Quanzhou Science and Technology Plan (No. 2022C015R). We thank Fucai Wang (Huaqiao University) and Lei Tong (Huaqiao University) for their technical assistance.

1. Peanut worm homogenate preparation
<ol>
	<li>Add 50 g of peanut worms and 150 mL of saline solution into the homogenizer.</li>
	<li>Homogenize at 24000 rpm for 60 s. 
	NOTE: Repeat 3 times.</li>
	<li>Centrifuge the homogenate at 9710 x g for 30 min at 4 &#176;C.</li>
	<li>Collect the supernatant as the peanut worm homogenate for further study.</li>
</ol>
2. Fibrinogen-PAGE gel preparation
<ol>
	<li>Weigh 0.01 g of fibrinogen into a 50 mL glass beaker.</li>
	<li>Add 1.9 mL of DDH2O, 1.3 mL of Tris-HCl (1.5 M, pH 8.8), and 0.05 mL of SDS (10%, w/v) into the beaker; mix well by pipetting.</li>
	<li>Place the beaker at 37 &#176;C for 30 min. 
	NOTE: This step is to dissolve the fibrinogen.</li>
	<li>Add 1.7 mL of acrylamide (30%, w/v), 0.05 mL of ammonium persulfate (APS; 10%, w/v), 0.002 mL of TEMED; mix well by pipetting. 
	NOTE: This is separating gel.</li>
	<li>Pour the separating gel into a 10-well gel mold.</li>
	<li>Add 2 mL of DDH2O into the gel mold; wait for 30 min.</li>
	<li>Remove the water thoroughly by turning the mold upside down.</li>
	<li>Add 1.4 mL of DDH2O, 0.25 mL of Tris-HCl (1.0 M, pH 6.8), 0.02 mL of SDS (10%, w/v), 0.33 mL of acrylamide (30%, w/v), 0.02 mL of APS (10%, w/v), 0.002 mL of tetramethylethylenediamine (TEMED) into the beaker; mix well by pipetting. 
	NOTE: This is loading gel.</li>
	<li>Pour the loading gel into the same gel mold as step 2.5; insert the comb immediately. 
	NOTE: The fibrinogen-PAGE gel used here was composed of the fibrinogen (0.2%) containing separating gel and loading gel. The concentration of fibrinogen can be adjusted as needed.</li>
</ol>
3. Electrophoresis
<ol>
	<li>Place the prepared fibrinogen-PAGE gel together with the glue mold into the electrophoresis tank; pour 1300 mL of electrophoresis solution (1.963 g of Tris, 12.22 g of glycine, 0.65 g of SDS, and 1300 mL of DDH2O) into the tank; take out the comb.</li>
	<li>Add 5 &#181;L of 5x non-denaturing loading buffer to the 20 &#181;L of sFE and 20 &#181;L of&#160;peanut worm homogenate; mix well by pipetting; load them into the prepared fibrinogen-PAGE gel. 
	NOTE: Do not boil the mixture. sFE is a fibrino(geno)lytic enzyme identified previously6,7,8 and is used as a positive control in this study. Peanut worm homogenate is the sample to be detected. Non-denaturing loading buffer without bromophenol blue, &#946;-mercaptoethanol, and SDS compared with the SDS-PAGE loading buffer. Each sample lane is separated with the 1x SDS-PAGE loading buffer to clearly detect the movement of the protein.</li>
	<li>Perform electrophoresis at 80 V for 30 min, or 120 V for 20 min.</li>
	<li>Remove the fibrinogen-PAGE gel from the glue mold.</li>
</ol>
4. Renaturation
<ol>
	<li>Transfer the fibrinogen-PAGE gel to a plastic box.</li>
	<li>Add 100 mL of Tris-HCl (0.05 M, pH 7.4) and 2 mL of Triton X-100 into the box. Place it on a shaker at 60-70 rpm for 10 min.</li>
	<li>Remove the wash buffer by pipetting. 
	NOTE: Repeat three times.</li>
</ol>
5. Incubation
<ol>
	<li>Add 50 mL of Tris-HCl (0.05 M, pH 7.4) into the plastic box.</li>
	<li>Incubate at 37 &#176;C for 4 h. 
	NOTE: Incubation time can be adjusted according to the strength of fibrinogenolytic activity.</li>
</ol>
6. Staining
<ol>
	<li>Remove the incubation buffer by pipetting.</li>
	<li>Add 50 mL of Coomassie Brillant Blue Staining solution into the box and place the box on a shaker at 60-70 rpm for 30 min.</li>
</ol>
7. Decolorization
<ol>
	<li>Remove the staining solution by pipetting.</li>
	<li>Add 50 mL of DDH2O into the box.</li>
	<li>Place the box on a shaker at 60-70 rpm for 30 min. 
	NOTE: DD H2O was used as the destaining solution here. The shaking time can be adjusted according to the strength of fibrinogenolytic activity.</li>
</ol>

Unveiling Fibrinogenolytic Agents in Peanut Worms with 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>

The authors have no conflicts of interest to disclose.

sFE is a novel fibrino(geno)lytic enzyme isolated from peanut worms by our team previously3,6,8,13. However, the identification processes of sFE were time- and labor-consuming, involving the fibrinogenolytic activity detection, protein components separation, and activity confirmation stages. As a simple method, fibrinogen plate assay is mostly used in the preliminary screening stage to check th...

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.

<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

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.

Author Spotlight: Time and Cost-Effective Fibrinogen-PAGE for Fibrinogenolytic Studies

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 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.

After electrophoresis, all the bands of the marker were clearly displayed. The 1x SDS-PAGE loading lanes only showed 10 kDa bands (bromophenol blue). The sFE and peanut worm samples did not show any observable bands (Figure 1). Although the bands of the samples are not visible, the performance of the marker and bromophenol blue indicated that the electrophoresis was successful, and the samples were separated according to their molecular weight.
Although the whole ...

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

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

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

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JoVE Journal

Biochemistry

416 visualizações.  Quanzhou First Hospital Affiliated to Fujian Medical University. Nossa pesquisa está focada no sistema fibrinolítico. Neste estudo, estamos tentando simplificar a massa para triagem de agentes fibrinogenolíticos em vermes do amendoim usando a técnica Fibrinogen-PAGE. Desenvolvemos um novo método Fibrinogen-PAGE combinando os métodos PAGE e Fibrinogen em placa.Comparado com as outras técnicas atualmente utilizadas, nosso protocolo consiste em um estudo de 10 anos separado e exibindo os genes fibrinogenolítico...

Vídeo: Autor em destaque: Fibrinogênio-PAGE de tempo e custo efetivo para estudos fibrinogenolíticos

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.

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.