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 aluminum plate. Place magnetic stirrer to the &#34;heavy&#34; (H)-chamber.</li>
	<li>Prepare the 3.5% (v/v) and 12.5% (v/v) acrylamide (AA) solutions in 15 mL conical centrifuge tubes for the separation gel gradient (see recipes in Table 1). To prevent untimely polymerization, keep the centrifuge tubes on ice while preparing the solutions. 
	CAUTION: Acrylamide is neurotoxic and carcinogenic, wear protective clothes and gloves.</li>
	<li>Add 5% APS and TEMED right before pipetting the solutions to the gradient mixer. Pipet the 12.5% solution to the H-chamber.
	<ol>
		<li>Remove air bubbles from the channel connecting the &#34;light&#34; (L) and H-chamber by opening the valve connecting the two chambers allowing solution to enter to the L-chamber. Close the valve and pipet the traces of solution back to H-chamber.</li>
		<li>Finally, pipet the 3.5% solution to the L-chamber.</li>
	</ol>
	</li>
	<li>Switch on the magnetic stirrer (the speed of the stir is not critical, but it should ensure proper mixing of the heavy and light solutions), open the valves and switch on the peristaltic pump. Allow the gel solutions to flow between the glass and aluminum plate, the flowrate should be roughly 0.5 mL/min. The needle must be above the liquid all the time, it can be attached to the upper part of the glass plate with a tape.</li>
	<li>When the H- and L-chambers have emptied, fill them with ultrapure water and allow water&#160;to gently overlay the gel surface. The gel polymerization takes around 1-2 hours at RT.</li>
	<li>Prepare the 3% acrylamide solution (see recipe in Table 1) for the stacking gel at RT. Pipet the stacking gel on top of the polymerized separation gel (before casting the stacking gel, remove the water overlaying the gel surface) and place a sample gel comb between the glass and aluminum plate avoiding air bubbles.
	<ol>
		<li>Allow to polymerize 30-60 min at RT. Remove the comb gently under ultrapure water. Store the gel at +4 &#730;C. 
		Pause point. The gel can be stored at +4 &#730;C for a few days. The gel should be kept in moist conditions to avoid drying of the wells and the gel surface.</li>
	</ol>
	</li>
</ol>
2. Thylakoid Solubilization1,2,3
NOTE: All steps should be performed under very dim light. Keep samples and buffers on ice.
<ol>
	<li>Dilute isolated thylakoids with ice-cold 25BTH20G buffer to a final chlorophyll concentration of 1 mg/mL. For 2D-BN-SDS-PAGE analysis roughly 4-8 &#181;g of chlorophyll/ sample is suitable. 
	NOTE: The thylakoids used in the experiments must be isolated from fresh leaves (for protocol of thylakoid isolation, see3)</li>
	<li>Add an equal volume of detergent buffer, i.e., 2% &#946;-DM (w/v) or 2% digitonin (w/v). Mix the detergent to the thylakoid sample gently with the pipet tip and avoid making air bubbles. The final concentration of the detergent is 1% and that of the thylakoids 0.5 mg Chl/mL.
	<ol>
		<li>Solubilize the thylakoids for 2 min on ice (&#946;-DM) or 10 minutes at RT with continuous gentle mixing on a rocker/shaker (digitonin). 
		NOTE: Digitonin and &#946;-DM are generally used for the solubilization of thylakoid protein complexes. If other non-ionic detergents are used, the detergent concentration and the solubilization time must be first optimized. Usually the detergent concentration range from 0.5%-5% (v/v). 
		CAUTION: Digitonin is toxic, wear protective clothes and gloves</li>
	</ol>
	</li>
	<li>Remove the insolubilized material by centrifugation at 18,000 x g for 20 min, at +4 &#730;C.</li>
	<li>Transfer the supernatant to a new 1.5 mL tube and add 1/10 (v/v) of CBB buffer to the sample. 
	NOTE: When the overall composition of the thylakoid membrane protein complexes is examined, determining the yield and the represented thylakoid domain of solubilized fraction is recommended. To determine the yield of the solubilized material, take 5 &#181;L of the supernatant to a new tube (before adding CBB) and measure the Chl content and Chl a/b ratio5.</li>
</ol>
3. BN-PAGE1,2,3
<ol>
	<li>Set up the gel to a vertical electrophoresis system (e.g., Hoefer SE 250). Fill the upper buffer chamber with blue cathode buffer (see Table 1) and pour anode buffer to the lower buffer chamber.</li>
	<li>Load thylakoid sample (e.g., 5 &#181;g of chlorophyll) into the wells.</li>
	<li>Start the electrophoresis and gradually increase the voltage: 75 V for 30 min, 100 V for 30 min, 125 V for 30 min, 150 V for 1 h and 175 V until the complexes have been separated completely. Run the gel at +4&#730;C, either using a cold room or adjusting the gel temperature with a cooling system. 
	NOTE: Change the blue cathode buffer to a clear cathode buffer when the sample front has migrated about one third of the gel.</li>
	<li>After the electrophoretic run, scan the gel with a photoscanner for image archiving.</li>
</ol>
4. 2D-SDS-PAGE
<ol>
	<li>Assemble the vertical electrophoresis system (gel size 16 cm x 20 cm). Use 1 mm spacers.</li>
	<li>Prepare standard SDS gel (12% acrylamide, 6 M Urea, see recipe in Table 2) with a 2D-comb (single large well for the strip and one standard well for molecular weight marker).</li>
	<li>Cut the lane from BN-gel and place it in a (5 mL) tube. Add 2 mL of Laemmli buffer (containing 5% &#946;-mercaptoethanol) and incubate the strip for 45 min with gentle shaking at RT.</li>
	<li>Place the lane, with a help of e.g., a spacer, on top of the gel avoiding air bubbles.</li>
	<li>Pipet 5 &#181;L of molecular weight marker on a narrow piece of filter paper and place the paper to the standard well.</li>
	<li>To seal the BN-gel strip and the marker paper, pour 0.5% agarose (in running buffer) on top of the gel strip and allow to solidify.</li>
	<li>Perform electrophoresis according to standard protocols. After the electrophoretic run, visualize the proteins with e.g., Sypro Ruby stain or silver staining according to6.</li>
</ol>

<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 authors have nothing to disclose.

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 membrane protein complexes, nonionic detergents are commonly used for their ability to preserve the complexes in their native form. Here, two commonly used detergents &#946;-DM and digitonin were applied. Dodecyl maltoside solubilizes individual protein complexes, whereas digitonin can be used for the analysis of larger protein complex assemblies10. The bulky structured digitonin is unable to fit to the tightly appressed grana partitions and therefore solubilizes only the non-appressed regions of the thylakoid membrane3,11. It is therefore suited for the analysis of stroma thylakoids and grana margins. However, when digitonin is used together with aminocaproic acid (ACA), the combination solubilizes the entire thylakoid membrane, including also the appressed grana thylakoids12. Through an unknown mechanism, ACA allows digitonin to have access to the partition gap between adjacent grana membrane layers. Importantly, digitonin preserves labile interactions between protein complexes and can therefore be used for the analysis of labile protein super and megacomplexes, which are most abundant in the non-appressed thylakoid regions8. It must be noted that detergents always interfere with some of the labile interactions between the protein complexes and therefore it is not possible to isolate completely intact network of protein complexes. Some of the complexes are dissociation products that have been disconnected from larger protein complex associations during thylakoid solubilization and the electrophoresis. The quality of the BN-PAGE separation depends not only on the sample preparation (protein complex solubilization), but also on the quality of the thylakoid isolation, which must be done from fresh leaves. If special care is not taken, the PSII-LHCII supercomplexes typically degrade during &#946;-DM solubilization.
BN-PAGE maintains the integrity of the solubilized protein complexes. The separation capacity of the BN gel depends on the acrylamide gradient, and the gradient should be optimized based on the protein complexes of interest. The pore-size of the polyacrylamide gel can be modified by changing the concentration gradient (total acrylamide concentration, T) or by adjusting the bis-acrylamide concentration (C) relative to the total amount of acrylamide monomers4. The BN-PA gel gradient used here is optimized and well suited for the analysis of large protein super- and megacomplexes3. Importantly, pore-size of the stacking gel must be large to allow all complexes to enter to the separation gel. After protein complex separation with BN-PAGE, the composition or structure of each individual protein complex band can be further analyzed. For structural analysis the protein complex of interest must be eluted from the gel and most labile complexes may be destroyed during the elution. Therefore, sucrose density gradient is more often used for the protein complex purification for structural analysis. For the analysis of spectroscopic properties of the protein complexes in the gel, the clear-native PAGE must be used instead of BN-PAGE, since the Coomassie dye interferes with such measurements. For analysis of the subunit composition of the protein complexes, a denaturing 2D-SDS-PAGE is described here. The subunits of each complex are separated in a vertical line and can be easily identified. It has to be noted that several proteins may be present in a single spot and the subunits in the same vertical line may belong to separate complexes co-migrating in BN-PAGE.

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>&lt;em&gt;n&lt;/em&gt;-dodecyl-&amp;beta;-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>&amp;beta;-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&#039;-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>&lt;br/&gt; 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), PSII m/Cyt b6f, loosely bound (L)-LHCII trimer (Figure 1A). The slightly stronger detergent, &#946;-DM, solubilizes the entire thylakoid membrane (horizontal gel on the top of Figure 1B), but is unable to preserve weak interactions between protein complexes. Thylakoid solubilization with &#946;-DM typically produces four PSII-LHCII supercomplexes (with different amount of LHCII antenna attached), PSII dimer (d) and PSI m, ATPase, PSII m and Cytb6f, M-LHCII, L-LHCII and LHCII monomer (Figure 1B). Unlike &#946;-DM, digitonin produces only minor amount of LHCII monomer. The protein complex pattern may differ depending on plant exposure to different light conditions and might also differ between mutant lines, since the protein complex interactions are dynamic and dependent on i.e., protein phosphorylation. SDS-PA gels below the native gel strips in Figure 1 A and B represent the polypeptide composition of each protein complex initially solubilized with digitonin or &#946;-DM.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58369/58369fig1.jpg" /> 
Figure 1. A two-dimensional BN-PAGE/SDS-PAGE of Arabidopsis thylakoid. Thylakoid protein complexes solubilized with (A) 1% digitonin and (B) 1% &#946;-DM and separated first by 1D-BN-PAGE (the lanes on top) and subsequently on 2D-SDS-PAGE to demonstrate the individual protein composition of each complex. Due to the incubation of BN-strips with denaturing Laemmli buffer, the protein subunits of each complex (in the BN strip) dissociate and are separated in a vertical line during the 2D-SDS-PAGE. The protein identification is based on mass spectrometry analysis presented in references7,8. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58369/58369fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Buffer</td>
			<td>Content </td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>3.5% (T) Acrylamide (AA) BN Separation gel </td>
			<td>48% AA, 1.5% bis-AA: 148 &#181;L 
			3xGel Buffer: 700 &#181;L 
			75% (w/v) glycerol: 140 &#181;L 
			Ultrapure H2O: 1092 &#181;L 
			5% APS: 15 &#181;L 
			TEMED 3 &#181;L</td>
			<td>NOTE: Acrylamide is neurotoxic. Add APS and TEMED immediately before use. The recipe is sufficient for casting one small BN gel.</td>
		</tr>
		<tr>
			<td>12.5% (T) Acrylamide (AA) BN Separation gel </td>
			<td>48% AA, 1,5% bis-AA: 530 &#181;L 
			3xGel Buffer: 700 &#181;L 
			75% (w/v) glycerol: 560 &#181;L 
			Ultrapure H2O: 290 &#181;L 
			5% APS: 11 &#181;L 
			TEMED 2&#181;L</td>
			<td>NOTE: Acrylamide is neurotoxic. Add APS and TEMED immediately before use. The recipe is sufficient for casting one small BN gel.</td>
		</tr>
		<tr>
			<td>3 % (T) Acrylamide (AA) BN Stacking gel </td>
			<td>20% AA, 5% bis-AA: 180 &#181;L 
			3xGel Buffer: 500 &#181;L 
			Ultrapure H2O: 800 &#181;L 
			5% APS: 30 &#181;L 
			TEMED 3 &#181;L</td>
			<td>NOTE: Acrylamide is neurotoxic. Add APS and TEMED immediately before use. The recipe is sufficient for one small BN gel.</td>
		</tr>
		<tr>
			<td>3x Gel buffer </td>
			<td>1.5M ACA 
			150mM BisTris/HCl (pH 7.0)</td>
			<td>Store at + 4 &#730;C.</td>
		</tr>
		<tr>
			<td>CBB buffer </td>
			<td>100 mM BisTris/HCl (pH 7.0) 0.5 M ACA 
			30% (w/v) sucrose 
			50 mg/ml Serva Blue G</td>
			<td>Store at + 4 &#730;C</td>
		</tr>
		<tr>
			<td>Anode buffer </td>
			<td>50 mM BisTris/HCl (pH 7.0)</td>
			<td>Store at + 4 &#730;C. Buffer can be prepared as 10x stock solution</td>
		</tr>
		<tr>
			<td>Cathode buffer </td>
			<td>50 mM Tricine 
			15 mM BisTris 
			0.01% Serva Blue G</td>
			<td>Store at + 4 &#730;C. Buffer can be prepared as 10x stock solution, add the dye to the 1x solution</td>
		</tr>
		<tr>
			<td>25BTH20G </td>
			<td>25 mM BisTris/HCl (pH 7.0) 
			20% (w/v) glycerol 
			0.25 mg/ml Pefabloc (add freshly) 
			10 mM NaF (add freshly)</td>
			<td>Buffer can be prepared as 2X stock solution (store at + 4 &#730;C) , but add Pefabloc and NaF freshly</td>
		</tr>
		<tr>
			<td>Detergent buffer </td>
			<td>2% &#946;-dodecyl maltoside/Digitonin (w/v) 
			25 mM BisTris/HCl (pH 7.0) 
			20% (w/v) glycerol and 
			0.25 mg/ml Pefabloc (add freshly from the stock solution) 
			10 mM NaF (add freshly)</td>
			<td>Detergents can be prepared as 5-10% stock solutions (in water). If other detergents are used, the final detergent concentration has to be optimized.</td>
		</tr>
	</tbody>
</table>
Table 1. Buffers and solutions for native gel electrophoresis
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Buffer</td>
			<td>Content </td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Laemmli buffer </td>
			<td>138 mM Tris/HCL pH 6.8 
			6M Urea 
			22.2 % (v/v) glycerol 
			4.4 % SDS</td>
			<td>Reference: 9</td>
		</tr>
		<tr>
			<td>12% Acrylamide, 6M Urea SDS Separation gel </td>
			<td>50% AA, 1,33% bis-AA: 10.5 mL 
			20% SDS: 0.7 mL 
			1.5 M Tris-HCl (pH 8.8): 8.05mL 
			Urea: 12.6 g 
			MQ-H2O: 6.16 mL 
			10% APS: 200 &#181;L 
			TEMED 28 &#181;L</td>
			<td>NOTE: Acrylamide is neurotoxic.The recipe is suitable for casting one big SDS-gel.</td>
		</tr>
		<tr>
			<td>6% Acrylamide, 6M Urea SDS Stacking gel </td>
			<td>50% AA, 1.33% bis-AA: 1.2 mL 
			20% SDS: 0.2 mL 
			0.5 M Tris-HCl (pH 6.8): 2.5 mL 
			Urea: 3.6 g 
			MQ-H2O: 3.45 mL 
			10% APS: 100 &#181;L 
			TEMED 10 &#181;L</td>
			<td>NOTE: Acrylamide is neurotoxic.</td>
		</tr>
		<tr>
			<td>SDS Running buffer </td>
			<td>19 mM Tris 
			2.5 mM Glycine 
			0.01 % SDS</td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 2. Buffers for 2D-SDS-PAGE

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 ...

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Nanyang Technological University

Research

JoVE Journal

Biochemistry

12.9K Views.  University of Turku. Deze methode kan helpen bij het beantwoorden van belangrijke vragen over de samenstelling en dynamische organisatie van de fotosynthetische eiwitcomplexen in plant thylakoid membraan. Het belangrijkste voordeel van deze techniek is dat het de analyse van functionele interacties tussen inheemse eiwitcomplexen mogelijk maakt en de plasticiteit van de eiwitcomplexe organisatie in wisselende omgevingsomstandigheden opheldert. Stel de gel caster acht centimeter bij 10 centimeter platen volgens de ...

Video: Analysis of Thylakoid Membrane Protein Complexes by Blue Native Gel Electrophoresis

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 processes. Affinity purification coupled to mass spectrometry (AP-MS) has become the method of choice for identifying protein-protein interactions. However, conventional AP-MS studies provide information on protein interactions, but the organizational information is lost. To address this issue, we developed a strategy to unravel the distinct functional assemblies a protein might be involved in, by resolving affinity-purified protein complexes prior to their characterization by mass spectrometry. Protein complexes isolated through affinity purification of a bait protein using an epitope tag and competitive elution are separated through blue native electrophoresis. Comparison of protein migration profiles through correlation profiling using quantitative mass spectrometry allows assignment of interacting proteins to distinct molecular entities. This method is able to resolve protein complexes of close molecular weights that might not be resolved by traditional chromatographic techniques such as gel filtration. With little more work than conventional AP-geLC-MS/MS, we demonstrate this strategy may in many cases be adequate for obtaining protein complex topological information concomitantly to identifying protein interactions.

Here we present protocols for affinity purification of protein complexes and their separation by blue native PAGE, followed by protein correlation profiling using label free quantitative mass spectrometry. This method is useful to resolve interactomes into distinct protein complexes.

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

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 ...

CN-GELFrEE - Clear Native Gel-eluted Liquid Fraction Entrapment Electrophoresis

Protein complexes perform an array of crucial cellular functions. Elucidating their non-covalent interactions and dynamics is paramount for understanding the role of complexes in biological systems. While the direct characterization of biomolecular assemblies has become increasingly important in recent years, native fractionation techniques that are compatible with downstream analysis techniques, including mass spectrometry, are necessary to further expand these studies. Nevertheless, the field lacks a high-throughput, wide-range, high-recovery separation method for native protein assemblies. Here, we present clear native gel-eluted liquid fraction entrapment electrophoresis (CN-GELFrEE), which is a novel separation modality for non-covalent protein assemblies. CN-GELFrEE separation performance was demonstrated by fractionating complexes extracted from mouse heart. Fractions were collected over 2 hr and displayed discrete bands ranging from ~30 to 500 kDa. A consistent pattern of increasing molecular weight bandwidths was observed, each ranging ~100 kDa. Further, subsequent reanalysis of native fractions via SDS-PAGE showed molecular-weight shifts consistent with the denaturation of protein complexes. Therefore, CN-GELFrEE was proved to offer the ability to perform high-resolution and high-recovery native separations on protein complexes from a large molecular weight range, providing fractions that are compatible with downstream protein analyses.

This protocol describes how to prepare and perform clear native gel-eluted liquid fraction entrapment electrophoresis (CN-GELFrEE), a native separation technique for non-covalent biomolecular assemblies and proteins from heterogeneous samples that is compatible with various downstream protein analysis techniques.

Protein complexes perform an array of crucial cellular functions. Elucidating their non-covalent interactions and dynamics is paramount for understanding ...

Chemistry

Separation of Spinach Thylakoid Protein Complexes by Native Green Gel Electrophoresis and Band Characterization using Time-Correlated Single Photon Counting

The light reactions of photosynthesis are carried out by a series of pigmented protein complexes in the thylakoid membranes. The stoichiometry and organization of these complexes is highly dynamic on both long and short time scales due to processes that adapt photosynthesis to changing environmental conditions (i.e., non-photochemical quenching, state transitions, and the long-term response). Historically, these processes have been described spectroscopically in terms of changes in chlorophyll fluorescence, and spectroscopy remains a vital method for monitoring photosynthetic parameters. There are a limited number of ways in which the underlying protein complex dynamics can be visualized. Here we describe a fast and simple method for the high-resolution separation and visualization of thylakoid complexes, native green gel electrophoresis. This method is coupled with time-correlated single photon counting for detailed characterization of the chlorophyll fluorescence properties of bands separated on the green gel.

Here we present a protocol to separate solubilized thylakoid complexes by Native Green Gel electrophoresis. Green gel bands are subsequently characterized by Time Correlated Single Photon Counting (TCSPC) and basic steps for data analysis are provided.

The light reactions of photosynthesis are carried out by a series of pigmented protein complexes in the thylakoid membranes. The stoichiometry and ...

Environment

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 ...

Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) for Analysis of Multiprotein Complexes from Cellular Lysates

Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other proteins (Sali, Glaeser et al. 2003). Thus, the study of protein-protein interaction networks requires the detailed characterization of MPCs to gain an integrative understanding of protein function and regulation. For identification and analysis, MPCs must be separated under native conditions. In this video, we describe the analysis of MPCs by blue native polyacrylamide gel electrophoresis (BN-PAGE). BN-PAGE is a technique that allows separation of MPCs in a native conformation with a higher resolution than offered by gel filtration or sucrose density ultracentrifugation, and is therefore useful to determine MPC size, composition, and relative abundance (Sch&auml;gger and von Jagow 1991); (Sch&auml;gger, Cramer et al. 1994). By this method, proteins are separated according to their hydrodynamic size and shape in a polyacrylamide matrix. Here, we demonstrate the analysis of MPCs of total cellular lysates, pointing out that lysate dialysis is the crucial step to make BN-PAGE applicable to these biological samples. Using a combination of first dimension BN- and second dimension SDS-PAGE, we show that MPCs separated by BN-PAGE can be further subdivided into their individual constituents by SDS-PAGE. Visualization of the MPC components upon gel separation is performed by standard immunoblotting. As an example for MPC analysis by BN-PAGE, we chose the well-characterized eukaryotic 19S, 20S, and 26S proteasomes.

Blue Native Polyacrylamide Gel Electrophoresis BN-PAGE for Analysis of Multiprotein Complexes from Cellular Lysates

In this video, we describe the characterization of multiprotein complexes (MPCs) by blue native polyacrylamide gel electrophoresis (BN-PAGE). In a first dimension, dialyzed cellular lysates are separated by BN-PAGE to identify individual MPCs. In a second dimension SDS-PAGE, MPCs of interest are further subdivided to analyze their constituents by immunoblotting.

Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other ...

Biology

A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry

The introduced protocol provides a tool for the analysis of multiprotein complexes in the thylakoid membrane, by revealing insights into complex composition under different conditions. In this protocol the approach is demonstrated by comparing the composition of the protein complex responsible for cyclic electron flow (CEF) in Chlamydomonas reinhardtii, isolated from genetically different strains. The procedure comprises the isolation of thylakoid membranes, followed by their separation into multiprotein complexes by sucrose density gradient centrifugation, SDS-PAGE, immunodetection and comparative, quantitative mass spectrometry (MS) based on differential metabolic labeling (14N/15N) of the analyzed strains. Detergent solubilized thylakoid membranes are loaded on sucrose density gradients at equal chlorophyll concentration. After ultracentrifugation, the gradients are separated into fractions, which are analyzed by mass-spectrometry based on equal volume. This approach allows the investigation of the composition within the gradient fractions and moreover to analyze the migration behavior of different proteins, especially focusing on ANR1, CAS, and PGRL1. Furthermore, this method is demonstrated by confirming the results with immunoblotting and additionally by supporting the findings from previous studies (the identification and PSI-dependent migration of proteins that were previously described to be part of the CEF-supercomplex such as PGRL1, FNR, and cyt f). Notably, this approach is applicable to address a broad range of questions for which this protocol can be adopted and e.g. used for comparative analyses of multiprotein complex composition isolated from distinct environmental conditions.

A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry

The described comparative, quantitative proteomic approach aims at obtaining insights into the composition of multiprotein complexes&#160;under different conditions and is demonstrated by comparing genetically different strains. For quantitative analysis equal volumes of different fractions from a sucrose density gradient are mixed and analyzed by mass spectrometry.

The introduced protocol provides a tool for the analysis of multiprotein complexes in the thylakoid membrane, by revealing insights into complex ...

Purification of Native Complexes for Structural Study Using a Tandem Affinity Tag Method

Affinity purification approaches have been successful in isolating native complexes for proteomic characterization. Structural heterogeneity and a degree of compositional heterogeneity of a complex do not usually impede progress in conducting such studies. In contrast, a complex intended for structural characterization should be purified in a state that is both compositionally and structurally homogeneous as well as at a higher concentration than required for proteomics. Recently, there have been significant advances in the application of electron microscopy for structure determination of large macromolecular complexes. This has heightened interest in approaches to purify native complexes of sufficient quality and quantity for structural determination by electron microscopy. The Tandem Affinity Purification (TAP) method has been optimized to extract and purify an 18-subunit, ~ 0.8 MDa ribonucleoprotein assembly from budding yeast (Saccharomyces cerevisiae) suitable for negative stain and electron cryo microscopy. Herein is detailed the modifications made to the TAP method, the rationale for making these changes, and the approaches taken to assay for a compositionally and structurally homogeneous complex.

The Tandem Affinity Purification (TAP) method has been used extensively to isolate native complexes from cellular extract, primarily eukaryotic, for proteomics. Here, we present a TAP method protocol optimized for purification of native complexes for structural studies.

Affinity purification approaches have been successful in isolating native complexes for proteomic characterization. Structural heterogeneity and a degree ...

Analysis of Mitochondrial Respiratory Chain Complexes in Cultured Human Cells using Blue Native Polyacrylamide Gel Electrophoresis and Immunoblotting

Mitochondrial respiration is performed by oxidative phosphorylation (OXPHOS) complexes within mitochondria. Internal and environmental factors can perturb the assembly and stability of OXPHOS complexes. This protocol describes the analysis of mitochondrial respiratory chain complexes by blue native polyacrylamide gel electrophoresis (BN-PAGE) in application to cultured human cells. First, mitochondria are extracted from the cells using digitonin, then using lauryl maltoside, the intact OXPHOS complexes are isolated from the mitochondrial membranes. The OXPHOS complexes are then resolved by gradient gel electrophoresis in the presence of the negatively charged dye, Coomassie blue, which prevents protein aggregation and ensures electrophoretic mobility of protein complexes towards the cathode. Finally, the OXPHOS complexes are detected by standard immunoblotting. Thus, BN-PAGE is a convenient and inexpensive technique that can be used to evaluate the assembly of entire OXPHOS complexes, in contrast to the basic SDS-PAGE allowing the study of only individual OXPHOS complex subunits.

This protocol demonstrates the analysis of mitochondrial respiratory chain complexes by blue native polyacrylamide gel electrophoresis. Here the method applied to cultured human cells is described.

Mitochondrial respiration is performed by oxidative phosphorylation (OXPHOS) complexes within mitochondria. Internal and environmental factors can perturb ...

Blue Native Polyacrylamide Gel Electrophoresis: A Non-Denaturing Separation Technique for Analysis of Intact Mitochondrial Respiratory Chain Complexes

Source: Konovalova, S. <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/59269">Analysis of Mitochondrial Respiratory Chain Complexes in Cultured Human Cells using Blue Native Polyacrylamide Gel Electrophoresis and Immunoblotting</a>. J. Vis. Exp. (2019)
In this video, we demonstrate an electrophoretic technique, called blue native PAGE, for the analysis of mitochondrial respiratory protein complexes in their natively folded and functionally active state. The anionic Coomassie blue dye causes the protein complexes to become negatively charged, allowing separation based on size and structure.

Source: Konovalova, S. Analysis of Mitochondrial Respiratory Chain Complexes in Cultured Human Cells using Blue Native Polyacrylamide Gel Electrophoresis ...

Analysis of Thylakoid Membrane Protein Complexes by Blue Native Gel Electrophoresis

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