Name: separation of spinach thylakoid protein complexes by native green gel electrophoresis and band characterization using time-correlated single photon counting
Uploaded: 2019-02-14T14:00:00
Description: Watch this Scientific Journal Video about Separation of Spinach Thylakoid Protein Complexes by Native Green Gel Electrophoresis and Band Characterization using Time-Correlated Single Photon Counting a

Funding and support were provided by the Department of Chemistry at Michigan State University.

1. Stock Solutions Preparation for Pouring Native Green Gels
<ol>
	<li>Prepare a 4x concentrated buffer solution for resolving gels consisting of 40% glycerol, 200 mM glycine, and 100 mM Tris buffered to pH 8.3.</li>
	<li>Prepare a 4x concentrated buffer solution for stacking gels consisting of 40% glycerol, 200 mM glycine, and 100 mM Tris buffered to pH 6.3.</li>
	<li>Store these buffers at 4 &#176;C to prevent the growth of mold. 
	Note: The buffers are stable for months at 4 &#176;C, so preparation of 100-200 m...

<ol>
	<li>Yamori, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photosynthetic+response+to+fluctuating+environments+and+photoprotective+strategies+under+abiotic+stress.">Photosynthetic response to fluctuating environments and photoprotective strategies under abiotic stress.</a> Journal of Plant Research. 129 (3), 379-395 (2016).</li><li>Kim, J. H., Nemson, J. A., Melis, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photosystem+II+reaction+center+damage+and+repair+in+Dunaliella+salina+(Green+Alga)+(analysis+under+physiological+and+irradiance-stress+conditions).">Photosystem II reaction center damage and repair in Dunaliella salina (Green Alga) (analysis under physiological and irradiance-stress conditions).</a> Plant Physiology. 103 (1), 181-189 (1993).</li><li>Sundby, C., McCaffery, S., Anderson, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Turnover+of+the+photosystem+II+D1+protein+in+higher+plants+under+photoinhibitory+and+nonphotoinhibitory+irradiance.">Turnover of the photosystem II D1 protein in higher plants under photoinhibitory and nonphotoinhibitory irradiance.</a> Journal of Biological Chemistry. 268 (34), 25476-25482 (1993).</li><li>Dietze, l. L., Br&#228;utigam, K., Pfannschmidt, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photosynthetic+acclimation:+state+transitions+and+adjustment+of+photosystem+stoichiometry-functional+relationships+between+short-term+and+longterm+light+quality+acclimation+in+plants.">Photosynthetic acclimation: state transitions and adjustment of photosystem stoichiometry-functional relationships between short-term and longterm light quality acclimation in plants.</a> FEBS Journal. 275 (6), 1080-1088 (2008).</li><li>Horton, P., Ruban, A. V., Walters, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+light+harvesting+in+green+plants.">Regulation of light harvesting in green plants.</a> Annual Review of Plant Physiology and Plant Molecular Biology. 47, 655-684 (1996).</li><li>Baker, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chlorophyll+fluorescence:+a+probe+of+photosynthesis+in+vivo.">Chlorophyll fluorescence: a probe of photosynthesis in vivo.</a> Annual Review of Plant Biology. 59, 89-113 (2008).</li><li>Williams, W. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+organization+and+interaction+of+the+two+photosystems+in+photosynthesis.">Spatial organization and interaction of the two photosystems in photosynthesis.</a> Nature. 225 (5239), 1214-1217 (1970).</li><li>Bellafiore, S., Barneche, F., Peltier, G., Rochaix, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=State+transitions+and+light+adaptation+require+chloroplast+thylakoid+protein+kinase+STN7.">State transitions and light adaptation require chloroplast thylakoid protein kinase STN7.</a> Nature. 433 (7028), 892-895 (2005).</li><li>Shapiguzov, A., 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=The+PPH1+phosphatase+is+specifically+involved+in+LHCII+dephosphorylation+and+state+transitions+in+Arabidopsis.">The PPH1 phosphatase is specifically involved in LHCII dephosphorylation and state transitions in Arabidopsis.</a> Proceedings of the National Academy of Sciences USA. 107 (10), 4782-4787 (2010).</li><li>Pribil, M., Pesaresi, P., Hertle, A., Barbato, R., Leister, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+plastid+protein+phosphatase+TAP38+in+LHCII+dephosphorylation+and+thylakoid+electron+flow.">Role of plastid protein phosphatase TAP38 in LHCII dephosphorylation and thylakoid electron flow.</a> Public Library of Science Biology. 8 (1), (2010).</li><li>Larsson, U. K., Jergil, B., Andersson, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+the+lateral+distribution+of+the+light-harvesting+chlorophyll-a/b-protein+complex+induced+by+its+phosphorylation.">Changes in the lateral distribution of the light-harvesting chlorophyll-a/b-protein complex induced by its phosphorylation.</a> European Journal of Biochemistry. 136 (1), 25-29 (1983).</li><li>Minagawa, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=State+transitions--the+molecular+remodeling+of+photosynthetic+supercomplexes+that+controls+energy+flow+in+the+chloroplast.">State transitions--the molecular remodeling of photosynthetic supercomplexes that controls energy flow in the chloroplast.</a> Biochimica et Biophysica Acta. 1807 (8), 897-905 (2011).</li><li>Ruban, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonphotochemical+Chlorophyll+Fluorescence+Quenching:+Mechanism+and+Effectiveness+in+Protecting+Plants+from+Photodamage.">Nonphotochemical Chlorophyll Fluorescence Quenching: Mechanism and Effectiveness in Protecting Plants from Photodamage.</a> Plant Physiology. 170 (4), 1903-1916 (2016).</li><li>Rokka, A., Suorsa, M., Saleem, A., Battchikova, N., 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=Synthesis+and+assembly+of+thylakoid+protein+complexes:+multiple+assembly+steps+of+photosystem+II.">Synthesis and assembly of thylakoid protein complexes: multiple assembly steps of photosystem II.</a> Biochemical Journal. 388 (Pt 1), 159-168 (2005).</li><li>Li, L., Aro, E. M., Millar, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+Photodamage+and+Protein+Turnover+in+Photoinhibition.">Mechanisms of Photodamage and Protein Turnover in Photoinhibition.</a> Trends in Plant Science. , (2018).</li><li>Dunahay, T. G., Staehelin, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+photosystem+I+complexes+from+octyl+glucoside/sodium+dodecyl+sulfate+solubilized+spinach+thylakoids:+characterization+and+reconstitution+into+liposomes.">Isolation of photosystem I complexes from octyl glucoside/sodium dodecyl sulfate solubilized spinach thylakoids: characterization and reconstitution into liposomes.</a> Plant Physiology. 78 (3), 606-613 (1985).</li><li>Allen, K. D., Staehelin, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resolution+of+16+to+20+chlorophyllprotein+complexes+using+a+low+ionic+strength+native+green+gel+system.">Resolution of 16 to 20 chlorophyllprotein complexes using a low ionic strength native green gel system.</a> Analytical Biochemistry. 194 (1), 214-222 (1991).</li><li>Peter, G. F., Thornber, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+composition+and+organization+of+higher+plant+photosystem+II+light-harvesting+pigment-proteins.">Biochemical composition and organization of higher plant photosystem II light-harvesting pigment-proteins.</a> Journal of Biological Chemistry. 266 (25), 16745-16754 (1991).</li><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>Schwarz, E. M., Tietz, S., Froehlich, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photosystem+I-LHCII+megacomplexes+respond+to+high+light+and+aging+in+plants.">Photosystem I-LHCII megacomplexes respond to high light and aging in plants.</a> Photosynthesis Research. 136 (1), 107-124 (2018).</li><li>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 (2), 207-214 (2011).</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 (5), 951-962 (2017).</li><li>Porra, R. J., Thompson, W. E., 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. 975 (3), 384-394 (1989).</li></ol>

A successful thylakoid solubilization and native gel run will result in the resolution of multiple distinct visible green bands on the gel without significant distortion or smearing of the bands. Overloading the gel, a high detergent concentration, an incorrect sample pH, undissolved material, running the gel too rapidly or at too high a temperature, and an improperly poured gel are all factors that may contribute to poorly resolved thylakoid complexes. While optimizing the conditions of the gel itself (e.g., ac...

Photosynthetic organisms must constantly adjust their physiology to changing environmental conditions to maximize their productivity and successfully compete with neighbors1. This is especially true of the machinery responsible for the light reactions of photosynthesis, as ambient light conditions can fluctuate by three orders of magnitude between shadows and full sunlight. Additionally, environmental factors such as drought, cold, or heat stress can reduce the availability of carbon dioxide for carbon fixation, which is the natural electron sink for the products of the light reactions. Plants must, therefore, harvest and utilize solar radiation as efficiently as possible while retaining the ability to dissipate excess light energy as necessary. While photooxidative damage still occurs routinely under all light conditions2,3, failure to manage absorbed excitation energy successfully can lead to the catastrophic cell damage and death. Several adaptive mechanisms exist that allow the photosynthetic apparatus to be tuned both to changes in prevailing environmental conditions and to transient fluctuations (i.e., over both long and short timescales)4. These include the long-term response (LTR) and non-photochemical quenching (NPQ). NPQ is itself considered to encompass at least three other component phenomena, including state transitions (qT), rapidly inducible energy quenching (qE), and photoinhibition (qI)5.
These processes were originally observed and defined largely in terms of spectroscopic phenomena [e.g., NPQ refers to a drop in observed chlorophyll fluorescence (quenching of chlorophyll fluorescence) that is not due to an increase in the rate of photochemistry]6. The term &#34;state transitions&#34; similarly refers to the observed change in the relative amount of fluorescence from PSI and PSII7. While the spectroscopic techniques that have made enumeration of these phenomena possible [in particular, pulse amplitude modulated (PAM) fluorescence spectroscopy] and continue to be a vital means for observing and dissecting photosynthetic processes in vivo, a great deal of biochemistry is required to elucidate the mechanisms underlying these spectroscopic observations. State transitions, for instance, involves a phosphorylation/dephosphorylation cycle of the LHCII proteins by the STN7 kinase and TAP38/PPH1 phosphatase, respectively8,9,10. This cycle adjusts the physical distribution of the LHCII antenna between the two photosystems by moving a portion of LHCII trimers from PSII to PSI, thereby changing the absorption cross section of the photosystems11,12. The qE component of NPQ rapidly converts excess excitation energy into heat through the actions of the violaxanthin/zeaxanthin epoxidation/de-epoxidation cycle and the PsbS protein. The exact role of PsbS in this process is still not fully understood13. The qI component of NPQ, photoinhibition, is generally ascribed to damage to the D1 protein of PSII. Restoration of full photosynthetic competence requires an elaborate repair process to fix damaged PSII photocenters. The PSII repair cycle involves the migration of PSII complexes out of the granal stacks, dismantling of the complexes, replacement of damaged D1 proteins, reassembly of the PSII complexes, and movement of PSII complexes back into the granal stacks14. The exact nature of photoinhibition and PSII photodamage remains a subject of intense scrutiny15.
The difficulty in studying phenomena like state transitions or PSII repair arises in part from the fact that there is not one simple way to visualize the mechanics of complex biochemical systems. The classic biochemical approach to understanding a process is to first separate its components so that they can be characterized in isolation. Native gel electrophoresis arose from successful efforts in the 1980s to separate and characterize the photosystem complexes from the thylakoid membranes with more preparative methods (namely sucrose gradient centrifugation and chromatography)16. The detergent systems developed to gently solubilize the native complexes from the thylakoid membranes were soon adapted to electrophoretic separation methods, most notably by Allen and Staehelin17 and and Peter and Thornber18, giving rise to native green gel electrophoresis. While representing only one out of a variety of techniques in the experimental arsenal, native PAGE has a number of attractive characteristics that have made it a widely employed method in photosynthesis research. Native PAGE is relatively fast and simple, requiring little specialized equipment, while providing high resolution separation of a large number of thylakoid complexes simultaneously. This makes native PAGE a convenient tool for studying thylakoid dynamics and, when combined with standard PAGE in the second dimension as well as a variety of detergent and buffer systems, a versatile system for finding and characterizing new thylakoid complexes.
That being said, native green gels have had a reputation for being an unreliable technique, especially in inexperienced hands, as it is easy to produce poor results consisting of fuzzy, smeary gels with few bands. This problem was solved, in part, with the introduction of blue-native PAGE19. The use of coommassie dye in the BN buffer system makes protein separation more robust. Therefore, BN-PAGE is often an easier and more reliable technique for a relative novice to set up and can provide high resolution separations of thylakoid complexes. For these reasons, BN-PAGE has become the method of choice for most work of this field. While BN-PAGE is generally slower to run than green gel electrophoresis, its main drawback is that the coommassie dye staining interferes with the identification of faint chlorophyll-containing bands, while also making downstream spectroscopic characterization problematic.
The biochemical information provided by native gels and 2D SDS-PAGE can be greatly strengthened when combined with data from spectroscopic techniques. Regardless of the system employed, a central problem with using native gels to identify complexes is that the identification can always be challenged (i.e., the proteins found in a band could always represent comigrating complexes or components, rather than a single physiologically authentic complex). Spectroscopic characterization provides biophysical information about the pigments in green gel bands and can be used to determine what types of complexes they are likely to contain. Chlorophyll fluorescence is especially useful in this regard due to the often dramatically different spectra and fluorescence lifetimes that are characteristic of different photosynthetic pigment-protein complexes. While simple steady-state 77K fluorescence spectra have historically been useful in confirming the identities of native gel complexes, modern time-correlated single photon counting (TCSPC) can provide much more information. TCSPC allows not only the characterization of complexes based on fluorescence lifetimes, but also makes possible the detailed description of energy transfer between spectral components within a complex. This kind of characterization is becoming increasingly necessary as the use of various native gel systems spreads and new putative complexes are discovered, allowing the identification of protein complexes to be better authenticated and providing new biophysical information about how these complexes work.
In this paper we provide a method that allows those having little or no experience with native gel electrophoresis to achieve high quality resolution of native thylakoid complexes for the purpose of investigating the mechanics of the light reactions of photosynthesis. This basic technique can then be augmented at the experimenter&#39;s discretion to improve results or extend applicability to other species. We then describe the process for subjecting native green gel bands to TCSPC, as well as some steps for basic analysis and presentation of the data provided by the technique. The coupling of native gel electrophoresis with TCSPC analysis extends the utility of these gel systems by providing authentication and biophysical characterization of protein complexes within the bands. The green gel system described here is based on that developed by Allen and Staehelin17 with some modifications and is the same as that used in Schwarz et al.20. This system is one of many but has specific features that are useful for this methodology. It is rapid enough so that thylakoid isolation, gel electrophoresis, and TCSPC analysis convenient can be performed in one day, obviating potential problems of sample storage and degradation. We also find that this method is robust in the hands of inexperienced users, while still providing results that range from good to superior, depending on the degree of optimization.
It is important to bear in mind that the complexes visualized on a native gel depend on both the detergent and buffer systems used, as well as on the biology of the organism under investigation. Different detergent and buffer systems preferentially separate different kinds of complexes, and a given photosynthetic organism will have different complexes from other organisms, not all of which will be present under any given circumstance. The system described here is particularly suited to the study of PSI megacomplexes, as described in Schwarz et al.20, but it falls on the more destabilizing end of the spectrum for those studying PSII megacomplexes. For a comprehensive study of the various detergent and buffer systems used in native gel electrophoresis of thylakoid proteins, it is recommended to review J&#228;rvi et al.21 and Rantala et al.22.

<table>
	<tbody>
		<tr>
			<td>Glycine</td>
			<td>Sigma</td>
			<td>G8898</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>Sigma</td>
			<td>#648310</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>Sigma</td>
			<td>L3771</td>
			<td></td>
		</tr>
		<tr>
			<td>Decyl Maltoside</td>
			<td>Sigma</td>
			<td>D7658</td>
			<td>n-decyl beta d maltopyranoside, not dodecyl maltoside or alpha decyl maltoside</td>
		</tr>
		<tr>
			<td>Octyl Glucoside</td>
			<td>Sigma</td>
			<td>O8001</td>
			<td></td>
		</tr>
		<tr>
			<td>Acrylamide</td>
			<td>BioRad</td>
			<td>161-0148</td>
			<td>37.5/1 C 40% solution</td>
		</tr>
		<tr>
			<td>TEMED</td>
			<td>BioRad</td>
			<td>161-0800</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium Persulfate</td>
			<td>BioRad</td>
			<td>161-0700</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Chloride</td>
			<td>Sigma</td>
			<td>M2670</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Sigma</td>
			<td>P9333</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Representative results for green gel electrophoresis are presented in Figure 1. Lane 1 provides an example of ideal results for green gel electrophoresis of spinach thylakoids, in which a maximum number of clear, sharp green bands are visible. These results are somewhat atypical, in part because not all of the bands seen in lane 1 are normally present in a given sample. Additional sample cleanup, in the form of chloroplast isolation before thylakoid solubiliz...

Watch this Scientific Journal Video about Separation of Spinach Thylakoid Protein Complexes by Native Green Gel Electrophoresis and Band Characterization using Time-Correlated Single Photon Counting a

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

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 ...

This method is useful for answering certain key questions in the field of photosynthesis research such as are putative thylakoid complexes physiologically real complexes with energetically connected components? The main advantage of the green gel and TCSPC protocol described here is that it's quick and robust. Samples can easily be processed and analyzed in a single day, making issues of storage and stability less of a concern.Though TCSPC can provide insight into the photosynthetic systems that we've studied here, it can also be applied to other systems ranging from chemical through physical and biological such as studies of fluorescent molecular motion in liquids, cells, or novel-structural motifs such as monomolecular layers and ionic liquids. To begin this procedure, prepare the needed stock solutions and green mini gels as outlined in the text protocol. Next, obtain some ice and dim the lights.Using a glass Dounce homogenizer, completely homogenize the spinach leaves in approximately one to two milliliters of TMK buffer. To create a simple filtering device, cut a delicate task wipe in half and fold it into quarters. Remove the plunger from a syringe, and then pack the folded task wipe into the bottom of the syringe.Pipette the leaf homogenate into the center of the task-wipe filter, then use the plunger to pass the homogenate through the filter and collect it in a 1.5-milliliter centrifuge tube. Centrifuge the homogenate at 5, 000 g and four degrees Celsius for 10 minutes to pellet the insoluble material, including the thylakoid membranes. Discard the supernatant and resuspend the pellet in one milliliter of TMK buffer.To extract chlorophyll from each sample, take a 50-microliter aliquot and transfer it into a 1.5-milliliter microcentrifuge tube. Add 950 microliters of methanol, cap the tube, and mix by inverting it several times. Centrifuge at 10, 000 g for 10 minutes to pellet the precipitated proteins.Using the chlorophyll concentration measurements as a guide, remove and discard some volume from each sample as necessary until each tube contains the same total amount of chlorophyll. Centrifuge at 5, 000 g for 10 minutes to re-pellet the thylakoid membranes. Remove and discard the supernatant, being careful to remove all of the supernatant without disturbing the pellet.First, thaw an aliquot of TMK 30%glycerol detergent solution. Invert several times to mix and keep the solution on ice. Add an appropriate volume of the detergent solution to dissolve the pellet and result in a final chlorophyll concentration of one milligram per milliliter.Pipette up and down repeatedly while being careful to avoid frothing in the sample. Then, keep the sample on ice to allow thylakoid samples to solubilize. After solubilization is complete, centrifuge the sample at 10, 000 g and four degrees Celsius for 10 minutes to pellet the insoluble material, resulting in a clear, dark green supernatant.After this, load 15 microliters of solubilized thylakoid supernatant directly onto each well of a previously prepared 1.5-millimeter native gel. Using 1X running buffer, begin running the native green gel at 100 volts. Place the entire gel tank on wet ice for the duration of the run to mitigate resistive heating.When the gel has finished running, remove it from the electrophoresis cell and rinse the running buffer off of the gel plates with distilled water. Remove the top plate from the gel and then rinse the gel with distilled water. Place the gel, which is still on the bottom glass plate, on ice;when the gel is not in use, cover it with plastic wrap and keep it in the dark to prevent it from drying out.Next, use a sharp scalpel or razor blade to excise each band of interest when ready to proceed with the TCSPC analysis. Make sure that the excised band contains no contaminating band material. For each complex being analyzed, use a fluorescent spectrometer to make a room-temperature fluorescent spectrum between 600 and 800 nanometers.Place masking tape on either end of the slides and fold it over several times to create spacers, which will allow the slides to be held together firmly without compressing the space between, then sandwich the gel slice between two glass microscopy slides. Add a small amount of water to the gel slice at the edge of the glass slides to create a smooth interface that will reduce signal scattering. Next, clamp the gel sandwich in the beam path so that the beam strikes the gel slice through the open edge of the plates.For each complex, collect 10, 000 total data points at regular intervals across the fluorescence emission spectrum. To analyze the data for a given complex, first normalize the peak height of each decay curve for all of the wavelengths collected, then tail match each decay curve to the steady state fluorescence spectrum of the complex as outlined in the text protocol. Representative results for the green gel electrophoresis provides an example of an ideal result for the analysis of spinach thylakoids, where a number of clear, sharp green bands are visible.Lanes 2 and 3 present more typical results from simple thylakoid isolation and electrophoresis on a non-gradient 5%acrylamide gel. Lanes 4, 5, and 6 provide an example of poor results due to increasing degrees of under solubilization of the thylakoid sample, while lane 7 demonstrates the results that can be achieved when Arabidopsis thylakoids are used instead of spinach. After the data curves of the TCSPC data have been set to the same time register, they can be normalized to the same peak height, which allows for the curves to be compared as a first analysis of the data.Peak-normalized decay curves from different complexes can then be overlaid with one another at a given wavelength, allowing the differences in behavior between the complexes to be visualized. Tail matching the decay curves allows for the construction of decay-associated spectra. As seen here, the decay-associated spectra for the LHCII is notable for the lack of dynamic features and the shape of the LHCII fluorescence spectrum remains the same as the signal decays over time.The decay of the fluorescence spectrum is also delayed, requiring 100 picoseconds to reach maximum fluorescence. This suggests that energy is not transferred between energetically distinct pigments within the complex as the fluorescence decays. The decay-associated spectra is then constructed for the Band 5 complex.Compared to the LHCII, the fluorescence from Band 5 decays much more rapidly, reaching maximum intensity in only 30 picoseconds and then decaying to less than 20%of the initial intensity after 500 picoseconds. While attempting this procedure, it's important to remember that interpretation of the TCSPC data will rely on additional biochemical information identifying the components of the complexes under study, for instance, by 2 DSDS-PAGE and Western Blotting or mass spectrometry.

separation-spinach-thylakoid-protein-complexes-native-green-gel

Research

JoVE Journal

Environment

Optimized Negative Staining: a High-throughput Protocol for Examining Small and Asymmetric Protein Structure by Electron Microscopy

Structural determination of proteins is rather challenging for proteins with molecular masses between 40 - 200 kDa. Considering that more than half of natural proteins have a molecular mass between 40 - 200 kDa1,2, a robust and high-throughput method with a nanometer resolution capability is needed. Negative staining (NS) electron microscopy (EM) is an easy, rapid, and qualitative approach which has frequently been used in research laboratories to examine protein structure and protein-protein interactions. Unfortunately, conventional NS protocols often generate structural artifacts on proteins, especially with lipoproteins that usually form presenting rouleaux artifacts. By using images of lipoproteins from cryo-electron microscopy (cryo-EM) as a standard, the key parameters in NS specimen preparation conditions were recently screened and reported as the optimized NS protocol (OpNS), a modified conventional NS protocol 3 . Artifacts like rouleaux can be greatly limited by OpNS, additionally providing high contrast along with reasonably high&#8208;resolution (near 1 nm) images of small and asymmetric proteins. These high-resolution and high contrast images are even favorable for an individual protein (a single object, no average) 3D reconstruction, such as a 160 kDa antibody, through the method of electron tomography4,5. Moreover, OpNS can be a high&#8208;throughput tool to examine hundreds of samples of small proteins. For example, the previously published mechanism of 53 kDa cholesteryl ester transfer protein (CETP) involved the screening and imaging of hundreds of samples 6. Considering cryo-EM rarely successfully images proteins less than 200 kDa has yet to publish any study involving screening over one hundred sample conditions, it is fair to call OpNS a high-throughput method for studying small proteins. Hopefully the OpNS protocol presented here can be a useful tool to push the boundaries of EM and accelerate EM studies into small protein structure, dynamics and mechanisms.

More than half of proteins are small proteins (molecular mass &#60;200 kDa) that are challenging for both electron microscope imaging and three-dimensional reconstructions. Optimized negative staining is a robust and high-throughput protocol to obtain high contrast and relatively high resolution (~1 nm) images of small asymmetric proteins or complexes under different physiological conditions.

Structural determination of proteins is rather challenging for proteins with molecular masses between 40 - 200 kDa. Considering that more than half of ...

Automated Gel Size Selection to Improve the Quality of Next-generation Sequencing Libraries Prepared from Environmental Water Samples

Next-generation sequencing of environmental samples can be challenging because of the variable DNA quantity and quality in these samples. High quality DNA libraries are needed for optimal results from next-generation sequencing. Environmental samples such as water may have low quality and quantities of DNA as well as contaminants that co-precipitate with DNA. The mechanical and enzymatic processes involved in extraction and library preparation may further damage the DNA. Gel size selection enables purification and recovery of DNA fragments of a defined size for sequencing applications. Nevertheless, this task is one of the most time-consuming steps in the DNA library preparation workflow. The protocol described here enables complete automation of agarose gel loading, electrophoretic analysis, and recovery of targeted DNA fragments. 
In this study, we describe a high-throughput approach to prepare high quality DNA libraries from freshwater samples that can be applied also to other environmental samples. We used an indirect approach to concentrate bacterial cells from environmental freshwater samples; DNA was extracted using a commercially available DNA extraction kit, and DNA libraries were prepared using a commercial transposon-based protocol. DNA fragments of 500 to 800 bp were gel size selected using Ranger Technology, an automated electrophoresis workstation. Sequencing of the size-selected DNA libraries demonstrated significant improvements to read length and quality of the sequencing reads.

This manuscript describes an automated gel size selection approach for purifying DNA fragments for next-generation sequencing. The Ranger Technology provides complete automation of the entire process of agarose gel loading, electrophoretic analysis, and recovery of targeted DNA fragments allowing for high-throughput and high quality next-generation sequencing libraries.

Next-generation sequencing of environmental samples can be challenging because of the variable DNA quantity and quality in these samples. High quality DNA ...

Characterization and Application of Passive Samplers for Monitoring of Pesticides in Water

Five different water passive samplers were calibrated under laboratory conditions for measurement of 124 legacy and current used pesticides. This study provides a protocol for the passive sampler preparation, calibration, extraction method and instrumental analysis. Sampling rates (RS) and passive sampler-water partition coefficients (KPW) were calculated for silicone rubber, polar organic chemical integrative sampler POCIS-A, POCIS-B, SDB-RPS and C18 disk. The uptake of the selected compounds depended on their physicochemical properties, i.e., silicone rubber showed a better uptake for more hydrophobic compounds (log octanol-water partition coefficient (KOW) &#62; 5.3), whereas POCIS-A, POCIS-B and SDB-RPS disk were more suitable for hydrophilic compounds (log KOW &#60; 0.70).

A protocol about the characterization and application of five different passive sampling devices is presented.

Five different water passive samplers were calibrated under laboratory conditions for measurement of 124 legacy and current used pesticides. This study ...

Extraction and Characterization of Surfactants from Atmospheric Aerosols

Surface-active compounds, or surfactants, present in atmospheric aerosols are expected to play important roles in the formation of liquid water clouds in the Earth&#39;s atmosphere, a central process in meteorology, hydrology, and for the climate system. But because specific extraction and characterization of these compounds have been lacking for decades, very little is known on their identity, properties, mode of action and origins, thus preventing the full understanding of cloud formation and its potential links with the Earth&#39;s ecosystems.
In this paper we present recently developed methods for 1) the targeted extraction of all the surfactants from atmospheric aerosol samples and for the determination of 2) their absolute concentrations in the aerosol phase and 3) their static surface tension curves in water, including their Critical Micelle Concentration (CMC). These methods have been validated with 9 references surfactants, including anionic, cationic and non-ionic ones. Examples of results are presented for surfactants found in fine aerosol particles (diameter &#60;1 &#956;m) collected at a coastal site in Croatia and suggestions for future improvements and other characterizations than those presented are discussed.

Methods are presented for the targeted extraction of surfactants present in atmospheric aerosols and the determination of their absolute concentrations and surface tension curves in water, including their Critical Micelle Concentration (CMC).

Surface-active compounds, or surfactants, present in atmospheric aerosols are expected to play important roles in the formation of liquid water clouds in ...

Measuring and Mapping Patterns of Soil Erosion and Deposition Related to Soil Carbonate Concentrations Under Agricultural Management

Spatial patterns of soil erosion and deposition can be inferred from differences in ground elevation mapped at appropriate time increments. Such changes in elevation are related to changes in near-surface soil carbonate (CaCO3) profiles. The objective is to describe a simple conceptual model and detailed protocol for repeatable field and laboratory measurements of these quantities. Here, accurate elevation is measured using a ground-based differential global positioning system (GPS); other data acquisition methods could be applied to the same basic method. Soil samples are collected from prescribed depth intervals and analyzed in the lab using an efficient and precise modified pressure-calcimeter method for quantitative analysis of inorganic carbon concentration. Standard statistical methods are applied to point data, and representative results show significant correlations between changes in soil surface layer CaCO3 and changes in elevation consistent with the conceptual model; CaCO3 generally decreased in depositional areas and increased in erosional areas. Maps are derived from point measurements of elevation and soil CaCO3 to aid analyses. A map of erosional and depositional patterns at the study site, a rain-fed winter wheat field cropped in alternating wheat-fallow strips, shows the interacting effects of water and wind erosion affected by management and topography. Alternative sampling methods and depth intervals are discussed and recommended for future work relating soil erosion and deposition to soil CaCO3.

Spatial patterns of soil erosion and deposition can be inferred from differences in ground elevation mapped at appropriate time increments. Such changes in elevation are related to changes in near-surface soil carbonates. Repeatable methods for field and laboratory measurements of these quantities and data analysis methods are described here.

Spatial patterns of soil erosion and deposition can be inferred from differences in ground elevation mapped at appropriate time increments. Such changes ...

Collection and Extraction of Occupational Air Samples for Analysis of Fungal DNA

Traditional methods of identifying fungal exposures in occupational environments, such as culture and microscopy-based approaches, have several limitations that have resulted in the exclusion of many species. Advances in the field over the last two decades have led occupational health researchers to turn to molecular-based approaches for identifying fungal hazards. These methods have resulted in the detection of many species within indoor and occupational environments that have not been detected using traditional methods. This protocol details an approach for determining fungal diversity within air samples through genomic DNA extraction, amplification, sequencing, and taxonomic identification of fungal internal transcribed spacer (ITS) regions. ITS sequencing results in the detection of many fungal species that are either not detected or difficult to identify to species level using culture or microscopy. While these methods do not provide quantitative measures of fungal burden, they offer a new approach to hazard identification and can be used to determine overall species richness and diversity within an occupational environment.

Determining the fungal diversity within an environment is a method utilized in occupational health studies to identify health hazards. This protocol describes DNA extraction from occupational air samples for amplification and sequencing of fungal ITS regions. This approach detects many fungal species that can be overlooked by traditional assessment methods.

Traditional methods of identifying fungal exposures in occupational environments, such as culture and microscopy-based approaches, have several ...

Characterization of Aquatic Biofilms with Flow Cytometry

Biofilms are dynamic consortia of microorganism that play a key role in freshwater ecosystems. By changing their community structure, biofilms respond quickly to environmental changes and can be thus used as indicators of water quality. Currently, biofilm assessment is mostly based on integrative and functional endpoints, such as photosynthetic or respiratory activity, which do not provide information on the biofilm community structure. Flow cytometry and computational visualization offer an alternative, sensitive, and easy-to-use method for assessment of the community composition, particularly of the photoautotrophic part of freshwater biofilms. It requires only basic sample preparation, after which the entire sample is run through the flow cytometer. The single-cell optical and fluorescent information is used for computational visualization and biological interpretation. Its main advantages over other methods are the speed of analysis and the high-information-content nature. Flow cytometry provides information on several cellular and biofilm traits in a single measurement: particle size, density, pigment content, abiotic content in the biofilm, and coarse taxonomic information. However, it does not provide information on biofilm composition on the species level. We see high potential in the use of the method for environmental monitoring of aquatic ecosystems and as an initial biofilm evaluation step that informs downstream detailed investigations by complementary and more detailed methods.

Flow cytometry in combination with visual clustering offers an easy-to-use and fast method for studying aquatic biofilms. It can be used for biofilm characterization, detection of changes in biofilm community structure, and detection of abiotic particles embedded in the biofilm.

Biofilms are dynamic consortia of microorganism that play a key role in freshwater ecosystems. By changing their community structure, biofilms respond ...

Quantifying Plant Soluble Protein and Digestible Carbohydrate Content, Using Corn (Zea mays) As an Exemplar

Elemental data are commonly used to infer plant quality as a resource to herbivores. However, the ubiquity of carbon in biomolecules, the presence of nitrogen-containing plant defensive compounds, and variation in species-specific correlations between nitrogen and plant protein content all limit the accuracy of these inferences. Additionally, research focused on plant and/or herbivore physiology require a level of accuracy that is not achieved using generalized correlations. The methods presented here offer researchers a clear and rapid protocol for directly measuring plant soluble proteins and digestible carbohydrates, the two plant macronutrients most closely tied to animal physiological performance. The protocols combine well characterized colorimetric assays with optimized plant-specific digestion steps to provide precise and reproducible results. Our analyses of different sweet corn tissues show that these assays have the sensitivity to detect variation in plant soluble protein and digestible carbohydrate content across multiple spatial scales. These include between-plant differences across growing regions and plant species or varieties, as well as within-plant differences in tissue type and even positional differences within the same tissue. Combining soluble protein and digestible carbohydrate content with elemental data also has the potential to provide new opportunities in plant biology to connect plant mineral nutrition with plant physiological processes. These analyses also help generate the soluble protein and digestible carbohydrate data needed to study nutritional ecology, plant-herbivore interactions and food-web dynamics, which will in turn enhance physiology and ecological research.

Quantifying Plant Soluble Protein and Digestible Carbohydrate Content, Using Corn Zea mays As an Exemplar

The protocols described herein provide a clear and approachable methodology for measuring soluble protein and digestible (non-structural) carbohydrate content in plant tissues. The ability to quantify these two plant macronutrients has significant implications for advancing the fields of plant physiology, nutritional ecology, plant-herbivore interactions and food-web ecology.

Elemental data are commonly used to infer plant quality as a resource to herbivores. However, the ubiquity of carbon in biomolecules, the presence of ...

Detection of Tilapia Lake Virus Using Conventional RT-PCR and SYBR Green RT-qPCR

The aim of this method is to facilitate the fast, sensitive and specific detection of Tilapia Lake Virus (TiLV) in tilapia tissues. This protocol can be used as part of surveillance programs, biosecurity measures and in TiLV basic research laboratories. The gold standard of virus diagnostics typically involves virus isolation followed by complementary techniques such as reverse-transcription polymerase chain reaction (RT-PCR) for further verification. This can be cumbersome, time-consuming and typically requires tissue samples heavily infected with virus. The use of RT-quantitative (q)PCR in the detection of viruses is advantageous because of its quantitative nature, high sensitivity, specificity, scalability and its rapid time to result. Here, the entire method of PCR based approaches for TiLV detection is described, from tilapia organ sectioning, total ribonucleic acid (RNA) extraction using a guanidium thiocyanate-phenol-chloroform solution, RNA quantification, followed by a two-step PCR protocol entailing, complementary deoxyribonucleic acid (cDNA) synthesis and detection of TiLV by either conventional PCR or quantitative identification via qPCR using SYBR green I dye. Conventional PCR requires post-PCR steps and will simply inform about the presence of the virus. The latter approach will allow for absolute quantification of TiLV down to as little as 2 copies and thus is exceptionally useful for TiLV diagnosis in sub-clinical cases. A detailed description of the two PCR approaches, representative results from two laboratories and a thorough discussion of the critical parameters of both have been included to ensure that researchers and diagnosticians find their most suitable and applicable method of TiLV detection.

This protocol diagnoses Tilapia Lake Virus (TiLV) in tilapia tissues using RT-PCR methodologies. The entire method is described from tissue dissection to total RNA extraction, followed by cDNA synthesis and detection of TiLV by either conventional PCR or quantitative PCR using dsDNA binding a fluorescent binding dye.

The aim of this method is to facilitate the fast, sensitive and specific detection of Tilapia Lake Virus (TiLV) in tilapia tissues. This protocol can be ...

Cereal Crop Ear Counting in Field Conditions Using Zenithal RGB Images

Ear density, or the number of ears per square meter (ears/m2), is a central focus in many cereal crop breeding programs, such as wheat and barley, representing an important agronomic yield component for estimating grain yield. Therefore, a quick, efficient, and standardized technique for assessing ear density would aid in improving agricultural management, providing improvements in preharvest yield predictions, or could even be used as a tool for crop breeding when it has been defined as a trait of importance. Not only are the current techniques for manual ear density assessments laborious and time-consuming, but they are also without any official standardized protocol, whether by linear meter, area quadrant, or an extrapolation based on plant ear density and plant counts postharvest. An automatic ear counting algorithm is presented in detail for estimating ear density with only sunlight illumination in field conditions based on zenithal (nadir) natural color (red, green, and blue [RGB]) digital images, allowing for high-throughput standardized measurements. Different field trials of durum wheat and barley distributed geographically across Spain during the 2014/2015 and 2015/2016 crop seasons in irrigated and rainfed trials were used to provide representative results. The three-phase protocol includes crop growth stage and field condition planning, image capture guidelines, and a computer algorithm of three steps: (i) a Laplacian frequency filter to remove low- and high-frequency artifacts, (ii) a median filter to reduce high noise, and (iii) segmentation and counting using local maxima peaks for the final count. Minor adjustments to the algorithm code must be made corresponding to the camera resolution, focal length, and distance between the camera and the crop canopy. The results demonstrate a high success rate (higher than 90%) and R2 values (of 0.62-0.75) between the algorithm counts and the manual image-based ear counts for both durum wheat and barley.

We present a protocol for counting durum wheat and barley ears, using natural color (RGB) digital photographs taken in natural sunlight under field conditions. With minimal adjustments for camera parameters and some environmental condition limitations, the technique provides precise and consistent results across a range of growth stages.