Name: analysis of thylakoid membrane protein complexes by blue native gel electrophoresis
Uploaded: 2018-09-28T14:30:00
Description: Watch this Scientific Journal Video about Analysis of Thylakoid Membrane Protein Complexes by Blue Native Gel Electrophoresis at JoVE.com

This research was financially supported by the Academy of Finland (project numbers 307335 and 303757) and Solar Energy into Biomass (SE2B) Marie Sk&#322;odowska-Curie grant agreement (675006). The protocol is based on reference3.

1. Preparing BN Gel1,2,3
<ol>
	<li>Set up the gel caster with 8 cm x 10 cm plates (rectangular glass and notched alumina plate) according to manufacturer&#39;s instructions using 0.75 mm spacers.</li>
	<li>Place a gradient mixer on a stir plate and connect it with the peristaltic pump by a tubing. Attach a syringe needle to the other end of the tubing and place the needle between the glass and...

<ol>
	<li>Sch&#228;gger, H., von Jagow, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blue+native+electrophoresis+for+isolation+of+membrane+protein+complexes+in+enzymatically+active+form.">Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form.</a> Analytical Biochemistry. 199, 223-231 (1991).</li><li>K&#252;gler, M., J&#228;nsch, L., Kruft, V., Schmitz, U. K., Braun, H. -. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+the+chloroplast+protein+complexes+by+blue-native+polyacrylamide+gel+electrophoresis+(BN-PAGE).">Analysis of the chloroplast protein complexes by blue-native polyacrylamide gel electrophoresis (BN-PAGE).</a> Photosynthesis Research. 53, 35-44 (1997).</li><li>J&#228;rvi, S., Suorsa, M., Paakkarinen, V., Aro, E. -. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+native+gel+systems+for+separation+of+thylakoid+protein+complexes:+novel+super-+and+mega-complexes.">Optimized native gel systems for separation of thylakoid protein complexes: novel super- and mega-complexes.</a> Biochemical Journal. 439, 207-214 (2011).</li><li>Strecker, V., Wumaier, Z., Wittig, I., Sch&#228;gger, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large+pore+gels+to+separate+mega+protein+complexes+larger+than+10+MDa+by+blue+native+electrophoresis:+Isolation+of+putative+respiratory+strings+or+patches.">Large pore gels to separate mega protein complexes larger than 10 MDa by blue native electrophoresis: Isolation of putative respiratory strings or patches.</a> Proteomics. 10, 3379-3387 (2010).</li><li>Porra, R. J., Thompson, W. A., Kriedemann, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+accurate+extinction+coefficients+and+simultaneous+equations+for+assaying+chlorophylls+a+and+b+extracted+with+four+different+solvents:+verification+of+the+concentration+of+chlorophyll+standards+by+atomic+absorption+spectroscopy.">Determination of accurate extinction coefficients and simultaneous equations for assaying chlorophylls a and b extracted with four different solvents: verification of the concentration of chlorophyll standards by atomic absorption spectroscopy.</a> Biochimica et Biophysica Acta (BBA) - Bioenergetics. , 384-394 (1989).</li><li>Blum, H., Beier, H., Gross, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+silver+staining+of+plant+proteins,+RNA+and+DNA+in+polyacrylamide+gels.">Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels.</a> Electrophoresis. 8, 93-99 (1987).</li><li>Aro, E. -. 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=Dynamics+of+photosystem+II:+a+proteomic+approach+to+thylakoid+protein+complexes.">Dynamics of photosystem II: a proteomic approach to thylakoid protein complexes.</a> Journal of Experimental Botany. 56, 347-356 (2005).</li><li>Suorsa, 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=Light+acclimation+involves+dynamic+re-organization+of+the+pigment-protein+megacomplexes+in+non-appressed+thylakoid+domains.">Light acclimation involves dynamic re-organization of the pigment-protein megacomplexes in non-appressed thylakoid domains.</a> The plant journal for cell and molecular biology. 84, 360-373 (2015).</li><li>Laemmli, U. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cleavage+of+Structural+Proteins+during+the+Assembly+of+the+Head+of+Bacteriophage+T4.">Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4.</a> Nature. 227, 680-685 (1970).</li><li>Sch&#228;gger, H., Pfeiffer, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Supercomplexes+in+the+respiratory+chains+of+yeast+and+mammalian+mitochondria.">Supercomplexes in the respiratory chains of yeast and mammalian mitochondria.</a> The EMBO Journal. 19, 1777-1783 (2000).</li><li>Rantala, S., Tikkanen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphorylation-induced+lateral+rearrangements+of+thylakoid+protein+complexes+upon+light+acclimation.">Phosphorylation-induced lateral rearrangements of thylakoid protein complexes upon light acclimation.</a> Plant Direct. 2, 1-12 (2018).</li><li>Rantala, M., Tikkanen, M., Aro, E. -. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+characterization+of+hierarchical+megacomplex+formation+in+Arabidopsis+thylakoid+membrane.">Proteomic characterization of hierarchical megacomplex formation in Arabidopsis thylakoid membrane.</a> Plant Journal. 92, 951-962 (2017).</li></ol>

The photosynthetic energy conversion machinery is composed of large multisubunit protein complexes, which are embedded in the thylakoid membrane. This protocol describes a basic method for analysis of the plant thylakoid protein complexes from Arabidopsis thaliana with BN-PAGE combined with 2D-SDS-PAGE. The protocol is also suitable for the analysis of thylakoid protein complexes from tobacco and spinach thylakoids, but might require small adjustments.
For the solubilization of membra...

Large multisubunit protein complexes photosystem PSI and PSII, Cyt b6f and ATPase coordinate the production of NADPH and ATP in photosynthetic light reactions. In higher plant chloroplasts, the complexes are located in the thylakoid membrane, which is a structurally heterogeneous membrane structure, comprising appressed grana and non-appressed stroma thylakoids. Blue native polyacrylamide gel electrophoresis (BN-PAGE) is an extensively used method in the analysis of large multisubunit protein complexes in their native and biologically active form. The method was established for the dissection of mitochondrial membrane protein complexes1, but has later been customized for the separation of protein complexes from the thylakoid membrane network3. The method is suitable (i) for the purification of individual thylakoid protein complexes for structural analysis, (ii) for determining native interactions between protein complexes and (iii) for the analysis of overall organization of the protein complexes upon changing environmental cues.
Prior to the separation, protein complexes are isolated from the membrane with carefully chosen nonionic detergents, which are generally mild and preserve the native structure of the protein complexes. Detergents contain hydrophobic and hydrophilic sites and form stable micelles above a certain concentration, called a critical micellar concentration (CMC). Increasing the detergent concentration above the CMC results in disruption of the lipid-lipid interactions and in the solubilization of protein complexes. The choice of detergent depends on the stability of the protein complex of interest and on the solubilization capacity of the detergent. Routinely used detergents include &#945;/&#946;-dodecyl-maltoside and digitonin. Following the solubilization of protein complexes in their native state, insoluble material is removed by centrifugation. In higher plants, the thylakoid membrane is highly heterogenic in structure and some detergents (e.g., digitonin) selectively solubilize only a specific fraction of the membrane3. Therefore, to characterize the protein complex organization or the interactions between the protein complexes, it is crucial to always determine the solubilization capacity of the chosen detergent by determining the chlorophyll content and the chlorophyll a/b ratio of supernatant to assess the yield and the represented thylakoid (sub)domain, respectively, of the solubilized fraction. The chlorophyll a/b ratio in intact thylakoids of growth-light acclimated plants is typically around 3, whereas the chl a/b value of thylakoid fractions enriched either in grana or stroma thylakoids falls below (~2.5) or exceeds (~4.5) the value of the total thylakoids, respectively.
To provide negative charge to the protein complexes, Coomassie brilliant blue (CBB) dye is added to the solubilized sample. Due to the charge shift, protein complexes migrate towards the anode and are separated on an acrylamide (AA) gradient according to their molecular mass and shape. Effective and high-resolution separation is achieved by using a linear acrylamide concentration gradient. During the electrophoresis, the protein complexes migrate towards the anode until they reach their size-dependent pore-size limit. The pore-size of polyacrylamide gel depends on (i) the total acrylamide/bis-acrylamide concentration (T) and (ii) on the cross-linker bis-acrylamide monomer concentration (C) relative to the total monomers4. After the separation with BN-PAGE, the protein complexes can be further subdivided into their individual protein subunits by second-dimension (2D)-SDS-PAGE. Here, we describe a detailed protocol for the analysis of thylakoid membrane protein complexes by BN-PAGE/2D-SDS-PAGE.

<table>
	<tbody>
		<tr>
			<td>6-aminocaproic acid (ACA)</td>
			<td>Sigma-Aldrich</td>
			<td>A2504</td>
			<td></td>
		</tr>
		<tr>
			<td>BisTris</td>
			<td>Sigma-Aldrich</td>
			<td>B4429</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>S0389</td>
			<td></td>
		</tr>
		<tr>
			<td>Acrylamide (AA)</td>
			<td>Sigma-Aldrich</td>
			<td>A9099</td>
			<td>Caution: Neurotoxic!</td>
		</tr>
		<tr>
			<td>n-dodecyl-&#946;-D-maltoside</td>
			<td>Sigma-Aldrich</td>
			<td>D4641</td>
			<td></td>
		</tr>
		<tr>
			<td>Tricine</td>
			<td>Sigma-Aldrich</td>
			<td>T0377</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Sigma-Aldrich</td>
			<td>T1503</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>VWR</td>
			<td>442444H</td>
			<td></td>
		</tr>
		<tr>
			<td>Urea</td>
			<td>VWR</td>
			<td>28877.292</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>J.T. Baker</td>
			<td>7044</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Fluoride (NaF)</td>
			<td>J.T. Baker</td>
			<td>3688</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA disodium salt</td>
			<td>J.T. Baker</td>
			<td>1073</td>
			<td></td>
		</tr>
		<tr>
			<td>Digitonin</td>
			<td>Calbiochem</td>
			<td>300410</td>
			<td>Caution:Toxic!</td>
		</tr>
		<tr>
			<td>Pefabloc SC</td>
			<td>Roche</td>
			<td>11585916001</td>
			<td></td>
		</tr>
		<tr>
			<td>Serva Coomassie Blue G</td>
			<td>Serva</td>
			<td>35050</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-mercaptoethanol</td>
			<td>Bio-Rad</td>
			<td>1610710</td>
			<td></td>
		</tr>
		<tr>
			<td>APS (Ammonium persulfate)</td>
			<td>Bio-Rad</td>
			<td>161-0700</td>
			<td></td>
		</tr>
		<tr>
			<td>TEMED (Tetramethylethylenediamine)</td>
			<td>Bio-Rad</td>
			<td>1610801</td>
			<td></td>
		</tr>
		<tr>
			<td>(N,N&#39;-Methylene)-Bis-Acrylamide</td>
			<td>Omnipur</td>
			<td>2610</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Fisher</td>
			<td>G0800</td>
			<td></td>
		</tr>
		<tr>
			<td>Prestained Protein Marker, Broad Range (7-175 kDa)</td>
			<td>New England Biolabs</td>
			<td>P7708</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon, Conical Centrifuge Tubes 15 ml</td>
			<td>Corning</td>
			<td>352093</td>
			<td></td>
		</tr>
		<tr>
			<td>Dual gel caster with 10 x 8 cm plates</td>
			<td>Hoefer</td>
			<td>SE215</td>
			<td></td>
		</tr>
		<tr>
			<td>Gradient maker SG5</td>
			<td>Hoefer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0.75 mm T-spacers</td>
			<td>Hoefer</td>
			<td>SE2119T-2-.75</td>
			<td></td>
		</tr>
		<tr>
			<td>Sample gel comb, 0.75 mm</td>
			<td>Hoefer</td>
			<td>SE211A-10-.75</td>
			<td></td>
		</tr>
		<tr>
			<td>Mighty Small SE250 vertical electrophoresis system</td>
			<td>Hoefer</td>
			<td>SE250</td>
			<td></td>
		</tr>
		<tr>
			<td>IPC-pump</td>
			<td>Ismatec</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Power supply, PowerPac HV</td>
			<td>Bio-Rad</td>
			<td>164-5097</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5424R</td>
			<td></td>
		</tr>
		<tr>
			<td>Rocker-Shaker</td>
			<td>Biosan</td>
			<td>BS-010130-AAI</td>
			<td></td>
		</tr>
		<tr>
			<td> 
			PROTEAN II xi Cell</td>
			<td>Bio-Rad</td>
			<td>1651813</td>
			<td></td>
		</tr>
	</tbody>
</table>

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 representative 2D-BN/SDS-PAGE system in Figure 1 demonstrates the separation of digitonin and &#946;-DM-solubilized thylakoid protein complexes and their detailed protein subunit composition. The protein complex pattern of digitonin solubilized thylakoids (horizontal gel on the top on the top of Figure 1A) contains the PSII-LHCII-PSI megacomplex, two large PSII-LHCII supercomplexes (sc), PSI-LHCII supercomplex, PSI monomer (m),...

Watch this Scientific Journal Video about Analysis of Thylakoid Membrane Protein Complexes by Blue Native Gel Electrophoresis at JoVE.com

Analysis of Thylakoid Membrane Protein Complexes by Blue Native Gel Electrophoresis

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 ...

This method can help answer key questions about the composition and dynamic organization of the photosynthetic protein complexes in plant thylakoid membrane. The main advantage of this technique is that it allows the analysis of functional interactions between native protein complexes and elucidates the plasticity of the protein complex organization in varying environmental conditions. Set up the gel caster eight-centimeter by 10-centimeter plates according to manufacturer's instructions using 0.75-millimeter spacers.Place a gradient mixer on a stir plate and connect it with the peristaltic pump by tubing. Attach a syringe needle to the other end of the tubing and place the needle between the glass and aluminum plate. Place a magnetic stirrer in the heavy chamber.Prepare 3.5%and 12.5%acrylamide solutions in 15-milliliter conical centrifuge tubes as detailed in the text protocol to use for the separation gel gradient. To prevent untimely polymerization keep the centrifuge tubes on ice while preparing the solutions. Add 5%APS and TEMED right before pippetting the solutions to the gradient mixer.Pipette the 12.5%solutions to the heavy chamber. Remove air bubbles from the channel connecting the light and heavy chamber by opening the valve connecting the two chambers allowing solution to enter to the light chamber. Close the valve and pipette the traces of solution back to the heavy chamber.Finally, pipette the 3.5%solution to the light chamber. Switch on the magnetic stirrer. Open the valves, and switch on the peristaltic pump.Allow the gel solutions to flow between the glass and aluminum plate at a flow rate of roughly 0.5 milliliters per minute. The needle must be above the liquid at all times. When the heavy and light chambers have emptied fill them with ultrapure water.And allow water to gently overlay the gel surface. Gel polymerization takes about one to two hours at room temperature. Prepare the 3%acrylamide solution for the stacking gel at room temperature.Pipette the stacking gel on top of the polymerized separation gel and place a sample gel comb between the glass and aluminum plate avoiding air bubbles. After allowing the gel to polymerize for 30 to 60 minutes at room temperature, remove the comb gently under ultrapure water. At this point the gel can be stored at positive four degrees Celsius in moist conditions for a few days.Dilute isolated thylakoids with ice-cold 25BTH20G buffer to a final chlorophyll concentration of one milligram per milliliter. Prepare two tubes for each thylakoid sample to perform both beta-DM and digitonin solubilization. Now add an equal volume of detergent buffer.Mix the detergent and thylakoid sample gently with a pipette and avoid making air bubbles. Solubilize the thylakoids for two minutes on ice for the beta-DM solubilization. Or 10 minutes at room temperature with continuous gentle mixing on a rocker or shaker for the digitonin solubilization.Following incubation, remove the insolubilized material by centrifugation at 18, 000 g for 20 minutes at positive four degrees Celsius. Transfer the supernatant to a new 1.5-milliliter tube and add 1/10 volume of Coomassie brilliant blue buffer to the sample. Set up the gel in a vertical electrophoresis system.Fill the upper buffer chamber with blue cathode buffer and pour anode buffer to the lower buffer chamber. Load the thylakoid samples into the wells. Start the electrophoresis and gradually increase the voltage as listed in the text protocol.Run the gel at positive four degrees Celsius either using a cold room or adjusting the gel temperature with a cooling system. Change the blue cathode buffer to a clear cathode buffer when the sample front has migrated about 1/3 of the gel. After the electrophoretic run scan the gel with a photo scanner for image archiving.To perform 2-SDS PAGE assemble the vertical electrophoresis system using one-millimeter spacers. Prepare a standard SDS gel with a 2-D comb as described in the text protocol. Cut the lane from the BN gel and place it in a five-milliliter tube.Then add two milliliters of Laemmli buffer and incubate the strip for 45 minutes with gentle shaking at room temperature. Place the lane with the help of a spacer on top of the gel avoiding air bubbles. Pipette five microliters of molecular weight marker on a narrow piece of filter paper and place the paper in the standard well.To seal the BN gel strip and the marker paper pour a 0.5%agarose in running buffer on top of the gel strip and allow it to solidify. Perform electrophoresis according to standard protocols. After the electrophoretic run visualize the proteins with SUPRO Ruby stain or silver staining.Representative results of thylakoid protein complex patterns of beta-DM and digitonin-solubilized samples are shown here. The small complexes migrate faster during the electrophoretic run and are therefore in the lower part of the gel. Whereas the large complexes are in the upper part of the gel.Digitonin generally preserves protein complexes in larger assemblies, while beta DM interferes with labile protein-protein interactions between the complexes. The subunit composition of protein complexes of beta-DM and digitonin-solubilized thylakoids are presented here. The subunits of each complex are found from the vertical line below the aligned complex.In principle, each spot in the 2-D gel represents an individual protein. But in practice, several proteins of same molecular mass may be present in a single spot. The identification of the spots is based on mass spectrometry analysis.After applying this procedure one can continue with other methods like electron microscopy and single particle analysis of the protein complexes eluted from the blue native gel in order to get structural insights into the protein complexes. To identify unknown protein subunits the individual protein spots from two-dimensional SDS PAGE can be analyzed with mass spec. Don't forget that working with acrylamide and digitonin can be extremely harmful and precautions such as wearing protective gloves should always be taken while performing this procedure.

Research

JoVE Journal

Biochemistry

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 ...

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological ...

Protein-tRNA Agarose Gel Retardation Assays for the Analysis of the N6-threonylcarbamoyladenosine TcdA Function

Protein-tRNA Agarose Gel Retardation Assays for the Analysis of the N6-threonylcarbamoyladenosine TcdA Function

We demonstrate methods for the expression and purification of tRNA(UUU) in Escherichia coli and the analysis by gel retardation assays of the binding of ...

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 ...

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 ...

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 ...

Split-BioID &#8212; Proteomic Analysis of Context-specific Protein Complexes in Their Native Cellular Environment

To complement existing affinity purification (AP) approaches for the identification of protein-protein interactions (PPI), enzymes have been introduced ...

2 in 1: One-step Affinity Purification for the Parallel Analysis of Protein-Protein and Protein-Metabolite Complexes

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of ...

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 ...