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.

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

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

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

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 ...

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417 Views.  Quanzhou First Hospital Affiliated to Fujian Medical University. Here, we present a fibrinogen-polyacrylamide gel electrophoresis (PAGE) protocol to rapidly separate and display the fibrinogenolytic agents of Sipunculus nudus.

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

Characterization of Multi-subunit Protein Complexes of Human MxA Using Non-denaturing Polyacrylamide Gel-electrophoresis

The formation of oligomeric complexes is a crucial prerequisite for the proper structure and function of many proteins. The interferon-induced antiviral effector protein MxA exerts a broad antiviral activity against many viruses. MxA is a dynamin-like GTPase and has the capacity to form oligomeric structures of higher order. However, whether oligomerization of MxA is required for its antiviral activity is an issue of debate. We describe here a simple protocol to assess the oligomeric state of endogenously or ectopically expressed MxA in the cytoplasmic fraction of human cell lines by non-denaturing polyacrylamide gel electrophoresis (PAGE) in combination with Western blot analysis. A critical step of the protocol is the choice of detergents to prevent aggregation and/or precipitation of proteins particularly associated with cellular membranes such as MxA, without interfering with its enzymatic activity. Another crucial aspect of the protocol is the irreversible protection of the free thiol groups of cysteine residues by iodoacetamide to prevent artificial interactions of the protein. This protocol is suitable for a simple assessment of the oligomeric state of MxA and furthermore allows a direct correlation of the antiviral activity of MxA interface mutants with their respective oligomeric states.

This article describes a simple and rapid protocol to evaluate the oligomeric state of the dynamin-like GTPase MxA protein from lysates of human cells using a combination of non-denaturing PAGE with western blot analysis.

The formation of oligomeric complexes is a crucial prerequisite for the proper structure and function of many proteins. The interferon-induced antiviral ...

Capillary Electrophoresis Separation of Monoclonal Antibody Isoforms Using a Neutral Capillary

Biotherapeutic proteins, such as monoclonal antibodies (mAbs), are feasible alternatives for the treatment of chronic-degenerative diseases. The biological activity of these proteins depends on their physicochemical properties. The use of high-performance techniques like chromatography and capillary electrophoresis has been described for the analysis of physicochemical heterogeneity of mAbs. Nowadays, capillary zone electrophoresis (CZE) technique constitutes one of the most resolutive and sensitive assays for the analysis of biomolecules. Besides, the electro-driven separation in CZE is governed by extensive properties of matter and offers the advantage of analyzing proteins close to their native state. However, the successful implementation of this technique for routine analysis depends on the skills of the analyst at the critical steps during sample and system preparation. The purpose of this tutorial is to detail the steps to succeed in the CZE analysis of mAbs. Further, this protocol can be used for the development and improvement of skills of the personnel involved in protein analytical chemistry laboratories.

Here, we present a comprehensive capillary zone electrophoresis protocol for the assessment of intrinsic physicochemical heterogeneity of monoclonal antibodies as a quality attribute.

Biotherapeutic proteins, such as monoclonal antibodies (mAbs), are feasible alternatives for the treatment of chronic-degenerative diseases. The ...

Analyzing Supercomplexes of the Mitochondrial Electron Transport Chain with Native Electrophoresis, In-gel Assays, and Electroelution

The mitochondrial electron transport chain (ETC) transduces the energy derived from the breakdown of various fuels into the bioenergetic currency of the cell, ATP. The ETC is composed of 5 massive protein complexes, which also assemble into supercomplexes called respirasomes (C-I, C-III, and C-IV) and synthasomes (C-V) that increase the efficiency of electron transport and ATP production. Various methods have been used for over 50 years to measure ETC function, but these protocols do not provide information on the assembly of individual complexes and supercomplexes. This protocol describes the technique of native gel polyacrylamide gel electrophoresis (PAGE), a method that was modified more than 20 years ago to study ETC complex structure. Native electrophoresis permits the separation of ETC complexes into their active forms, and these complexes can then be studied using immunoblotting, in-gel assays (IGA), and purification by electroelution. By combining the results of native gel PAGE with those of other mitochondrial assays, it is possible to obtain a completer picture of ETC activity, its dynamic assembly and disassembly, and how this regulates mitochondrial structure and function. This work will also discuss limitations of these techniques. In summary, the technique of native PAGE, followed by immunoblotting, IGA, and electroelution, presented below, is a powerful way to investigate the functionality and composition of mitochondrial ETC supercomplexes.

This protocol describes the separation of functional mitochondrial electron transport chain complexes (Cx) I-V and supercomplexes thereof using native electrophoresis to reveal information about their assembly and structure. The native gel can be subjected to immunoblotting, in-gel assays, and purification by electroelution to further characterize individual complexes.

The mitochondrial electron transport chain (ETC) transduces the energy derived from the breakdown of various fuels into the bioenergetic currency of the ...

Horizontal Gel Electrophoresis for Enhanced Detection of Protein-RNA Complexes

Native polyacrylamide gel electrophoresis is a fundamental tool of molecular biology that has been used extensively for the biochemical analysis of RNA-protein interactions. These interactions have been traditionally analyzed with polyacrylamide gels generated between two glass plates and samples electrophoresed vertically. However, polyacrylamide gels cast in trays and electrophoresed horizontally offers several advantages. For example, horizontal gels used to analyze complexes between fluorescent RNA substrates and specific proteins can be imaged multiple times as electrophoresis progresses. This provides the unique opportunity to monitor RNA-protein complexes at several points during the experiment. In addition, horizontal gel electrophoresis makes it possible to analyze many samples in parallel. This can greatly facilitate time course experiments as well as analyzing multiple reactions simultaneously to compare different components and conditions. Here we provide a detailed protocol for generating and using horizontal native gel electrophoresis for analyzing RNA-Protein interactions.

Native polyacrylamide gel electrophoresis is a fundamental tool for analyzing RNA-protein interactions. Traditionally most experiments have used vertical gels. However, horizontal gels provide several advantages, such as the opportunity to monitor complexes during electrophoresis. We provide a detailed protocol for generating and using horizontal native gel electrophoresis.

Native polyacrylamide gel electrophoresis is a fundamental tool of molecular biology that has been used extensively for the biochemical analysis of ...

DNA Polymerase Activity Assay Using Near-infrared Fluorescent Labeled DNA Visualized by Acrylamide Gel Electrophoresis

For any enzyme, robust, quantitative methods are required for characterization of both native and engineered enzymes. For DNA polymerases, DNA synthesis can be characterized using an in vitro DNA synthesis assay followed by polyacrylamide gel electrophoresis. The goal of this assay is to quantify synthesis of both natural DNA and modified DNA (M-DNA). These approaches are particularly useful for resolving oligonucleotides with single nucleotide resolution, enabling observation of individual steps during enzymatic oligonucleotide synthesis. These methods have been applied to the evaluation of an array of biochemical and biophysical properties such as the measurement of steady-state rate constants of individual steps of DNA synthesis, the error rate of DNA synthesis, and DNA binding affinity. By using modified components including, but not limited to, modified nucleoside triphosphates (NTP), M-DNA, and/or mutant DNA polymerases, the relative utility of substrate-DNA polymerase pairs can be effectively evaluated. Here, we detail the assay itself, including the changes that must be made to accommodate nontraditional primer DNA labeling strategies such as near-infrared fluorescently labeled DNA. Additionally, we have detailed crucial technical steps for acrylamide gel pouring and running, which can often be technically challenging.

This protocol describes the characterization of DNA polymerase synthesis of modified DNA through observation of changes to near-infrared fluorescently labeled DNA using gel electrophoresis and gel imaging. Acrylamide gels are used for high resolution imaging of the separation of short nucleic acids, which migrate at different rates depending on size.

For any enzyme, robust, quantitative methods are required for characterization of both native and engineered enzymes. For DNA polymerases, DNA synthesis ...

Use of Two Dimensional Semi-denaturing Detergent Agarose Gel Electrophoresis to Confirm Size Heterogeneity of Amyloid or Amyloid-like Fibers

Amyloid or amyloid-like fibers have been associated with many human diseases, and are now being discovered to be important for many signaling pathways. The ability to readily detect the formation of these fibers under various experimental conditions is essential for understanding their potential function. Many methods have been used to detect the fibers, but not without some drawbacks. For example, electron microscopy (EM), or staining with Congo Red or Thioflavin T often requires purification of the fibers. On the other hand, semi-denaturing detergent agarose gel electrophoresis (SDD-AGE) allows detection of the SDS-resistant amyloid-like fibers in the cell extracts without purification. In addition, it allows the comparison of the size difference of the fibers. More importantly, it can be used to identify specific proteins within the fibers by Western blotting. It is less time consuming and more easily accessible to a wider number of labs. SDD-AGE results often show variable degree of heterogeneity. It raises the question whether part of the heterogeneity results from the dissociation of the protein complex during the electrophoresis in the presence of SDS. For this reason, we have employed a second dimension of SDD-AGE to determine if the size heterogeneity seen in SDD-AGE is truly a result of fiber heterogeneity in vivo and not a result of either degradation or dissociation of some of the proteins during electrophoresis. This method allows fast, qualitative confirmation that the amyloid or amyloid-like fibers are not partially dissociating during the SDD-AGE process.

Here we use two-dimensional semi-denaturing agarose gel electrophoresis to confirm the presence of amyloid-like fibers of heterogeneous size and exclude the possibility that the size heterogeneity is due to dissociation of the amyloid fibers during the gel running process.

Amyloid or amyloid-like fibers have been associated with many human diseases, and are now being discovered to be important for many signaling pathways. ...

Analysis of AtHIRD11 Intrinsic Disorder and Binding Towards Metal Ions by Capillary Gel Electrophoresis and Affinity Capillary Electrophoresis

Plants are strongly dependent on their environment. In order to adjust to stressful changes (e.g., drought and high salinity), higher plants evolve classes of intrinsically disordered proteins (IDPs) to reduce oxidative and osmotic stress. This article uses a combination of capillary gel electrophoresis (CGE) and mobility shift affinity electrophoresis (ACE) in order to describe the binding behavior of different conformers of the IDP AtHIRD11 from Arabidopsis thaliana. CGE is used to confirm the purity of AtHIRD11 and to exclude fragments, posttranslational modifications, and other impurities as reasons for complex peak patterns. In this part of the experiment, the different sample components are separated by a viscous gel inside a capillary by their different masses and detected with a diode array detector. Afterward, the binding behavior of the sample towards various metal ions is investigated by ACE. In this case, the ligand is added to the buffer solution and the shift in migration time is measured in order to determine whether a binding event has occurred or not. One of the advantages of using the combination of CGE and ACE to determine the binding behavior of an IDP is the possibility to automate the gel electrophoresis and the binding assay. Furthermore, CGE shows a lower limit of detection than the classical gel electrophoresis and ACE is able to determine the manner of binding a ligand in a fast manner. In addition, ACE can also be applied to other charged species than metal ions. However, the use of this method for binding experiments is limited in its ability to determine the number of binding sites. Nevertheless, the combination of CGE and ACE can be adapted for characterizing the binding behavior of any protein sample towards numerous charged ligands.

This protocol combines the characterization of a protein sample by capillary gel electrophoresis and a fast-binding screening for charged ligands by affinity capillary electrophoresis. It is recommended for proteins with a flexible structure, such as intrinsically disordered proteins, to determine any differences in binding for different conformers.

Plants are strongly dependent on their environment. In order to adjust to stressful changes (e.g., drought and high salinity), higher plants evolve ...

Analysis of Thylakoid Membrane Protein Complexes by Blue Native Gel Electrophoresis

Photosynthetic electron transfer chain (ETC) converts solar energy to chemical energy in the form of NADPH and ATP. Four large protein complexes embedded in the thylakoid membrane harvest solar energy to drive electrons from water to NADP+ via two photosystems, and use the created proton gradient for production of ATP. Photosystem PSII, PSI, cytochrome b6f (Cyt b6f) and ATPase are all multiprotein complexes with distinct orientation and dynamics in the thylakoid membrane. Valuable information about the composition and interactions of the protein complexes in the thylakoid membrane can be obtained by solubilizing the complexes from the membrane integrity by mild detergents followed by native gel electrophoretic separation of the complexes. Blue native polyacrylamide gel electrophoresis (BN-PAGE) is an analytical method used for the separation of protein complexes in their native and functional form. The method can be used for protein complex purification for more detailed structural analysis, but it also provides a tool to dissect the dynamic interactions between the protein complexes. The method was developed for the analysis of mitochondrial respiratory protein complexes, but has since been optimized and improved for the dissection of the thylakoid protein complexes. Here, we provide a detailed up-to-date protocol for analysis of labile photosynthetic protein complexes and their interactions in Arabidopsis thaliana.

A protocol for the elucidation of plant thylakoid protein complex organization and composition with blue native polyacrylamide gel electrophoresis (BN-PAGE) and 2D-SDS-PAGE is described. The protocol is optimized for Arabidopsis thaliana, but can be used for other plant species with minor modifications.

Photosynthetic electron transfer chain (ETC) converts solar energy to chemical energy in the form of NADPH and ATP. Four large protein complexes embedded ...

Hybrid Clear/Blue Native Electrophoresis for the Separation and Analysis of Mitochondrial Respiratory Chain Supercomplexes

Complexes of the oxidative phosphorylation machinery form supramolecular protein arrangements named supercomplexes (SCs), which are believed to confer structural and functional advantages to mitochondria. SCs have been identified in many species, from yeast to mammal, and an increasing number of studies report disruption of their organization in genetic and acquired human diseases. As a result, an increasing number of laboratories are interested in analyzing SCs, which can be methodologically challenging. This article presents an optimized protocol that combines the advantages of Blue- and Clear-Native PAGE methods to resolve and analyze SCs in a time-effective manner. With this hybrid CN/BN-PAGE method, mitochondrial SCs extracted with optimal amounts of the mild detergent digitonin are exposed briefly to the anionic dye Coomassie Blue (CB) at the beginning of the electrophoresis, without exposure to other detergents. This short exposure to CB allows to separate and resolve SCs as effectively as with traditional BN-PAGE methods, while avoiding the negative impact of high CB levels on in-gel activity assays, and labile protein-protein interactions within SCs. With this protocol it is thus possible to combine precise and rapid in gel activity measurements with analytical techniques involving 2D electrophoresis, immuno-detection, and/or proteomics for advanced analysis of SCs.

Here we present a protocol to extract, resolve and identify mitochondrial supercomplexes which minimizes exposure to detergents and Coomassie Blue. This protocol offers an optimal balance between resolution, and preservation of enzyme activities, while minimizing the risk of losing labile protein-protein interactions.

Complexes of the oxidative phosphorylation machinery form supramolecular protein arrangements named supercomplexes (SCs), which are believed to confer ...

Rapid and Cost-Effective RNA Extraction of Rat Pancreatic Tissue

Regardless of the extraction method, optimized RNA extraction of tissues and cell lines are carried out in four stages: 1) homogenization, 2) effective denaturation of proteins from RNA, 3) ribonuclease inactivation, and 4) removal of contamination from DNA, proteins, and carbohydrates. However, it is very laborious to maintain the integrity of RNA when there are high levels of RNase in the tissue. Spontaneous autolysis makes it very difficult to extract RNA from pancreatic tissue without damaging it. Thus, a practical RNA extraction method is needed to maintain the integrity of pancreatic tissues during the extraction process. An experimental and comparative study of existing protocols was carried out by obtaining 20-30 mg of rat pancreatic tissues in less than 2 minutes and extracting the RNA. The results were assessed by electrophoresis. The experiments were carried out three times for generalization of the results. Immersing pancreatic tissue in RNA stabilization reagent at -80 &#176;C for 24 h yielded high integrity RNA, when the RNA extraction reagent was used as the reagent. The results obtained were comparable to the results obtained from commercial kits with spin column bindings.

The purity and integrity of the isolated RNA is a vital step in RNA dependent assays. Here, we present a practical, rapid, and inexpensive method to extract RNA from a small quantity of undamaged pancreatic tissue.

Regardless of the extraction method, optimized RNA extraction of tissues and cell lines are carried out in four stages: 1) homogenization, 2) effective ...