Name: metabolic labeling and membrane fractionation for comparative proteomic analysis of arabidopsis thaliana suspension cell cultures
Uploaded: 2013-09-28T13:00:00
Description: Watch this Scientific Journal Video about Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures at JoVE.com

PROCEDURE
Common reagents and buffers used in the extraction protocol:
<ol>
	<li>JPL medium for Arabidopsis thaliana suspension cell cultures
	<ul>
		<li>3 &mu;M H3BO3</li>
		<li>3 &mu;M MnSO4 x H2O</li>
		<li>1.1 &mu;M ZnSO4 x 7 H2O</li>
		<li>0.15 &mu;M KJ</li>
		<li>0.03 &mu;M Na2MoO4 x 2 H2O</li>
		<li>3 nM CoCl2 x 6 H2O</li>
		<li>3 nM CuSO4 x 5 H<s...

<ol>
	<li>Alexandersson, E., Saalbach, G., Larsson, C., Kjellbom, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arabidopsis+plasma+membrane+proteomics+identifies+components+of+transport,+signal+transduction+and+membrane+trafficking.">Arabidopsis plasma membrane proteomics identifies components of transport, signal transduction and membrane trafficking.</a> Plant Cell Physiol. 45, 1543-1556 (2004).</li><li>Brown, D. A., Rose, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sorting+of+GPI-anchored+proteins+to+glycolipid-enriched+membrane+subdomains+during+transport+to+the+apical+cell+surface.">Sorting of GPI-anchored proteins to glycolipid-enriched membrane subdomains during transport to the apical cell surface.</a> Cell. 68, 533-544 (1992).</li><li>Lanquar, V., 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=15N-metabolic+labeling+for+comparative+plasma+membrane+proteomics+in+Arabidopsis+cells.">15N-metabolic labeling for comparative plasma membrane proteomics in Arabidopsis cells.</a> Proteomics. 7, 750-754 (2007).</li><li>Kierszniowska, S., Walther, D., Schulze, W. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ratio-dependent+significance+thresholds+in+reciprocal+15N-labeling+experiments+as+a+robust+tool+in+detection+candidate+proteins+responding+to+biological+treatment.">Ratio-dependent significance thresholds in reciprocal 15N-labeling experiments as a robust tool in detection candidate proteins responding to biological treatment.</a> Proteomics. 9, 1916-1924 (2009).</li><li>Singer, S. J., Nicolson, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fluid+mosaic+model+of+the+structure+of+cell+membranes.">The fluid mosaic model of the structure of cell membranes.</a> Science. 175, 720-731 (1972).</li><li>Simons, K., Ikonen, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+rafts+in+cell+membranes.">Functional rafts in cell membranes.</a> Nature. 387, 569-5672 (1997).</li><li>Baumgart, T., 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=Large-scale+fluid/fluid+phase+separation+of+proteins+and+lipids+in+giant+plasma+membrane+vesicles.">Large-scale fluid/fluid phase separation of proteins and lipids in giant plasma membrane vesicles.</a> Proc. Natl. Acad. Sci. USA. 104, 3165-3170 (2007).</li><li>Silvius, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Partitioning+of+membrane+molecules+between+raft-and+non+raft-domains:+insights+from+model+membrane+studies.">Partitioning of membrane molecules between raft-and non raft-domains: insights from model membrane studies.</a> Biochim. Biophys. Acta. 1746, 193-202 (2005).</li><li>Tanner, W., Malinsky, J., Opekarova, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+plant+and+animal+cells,+detergent-resistant+membranes+do+not+define+functional+membrane+rafts.">In plant and animal cells, detergent-resistant membranes do not define functional membrane rafts.</a> Plant Cell. , (2011).</li><li>Lauwers, E., Andre, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+of+yeast+transporters+with+detergent-resistant+membranes+correlates+with+their+cell-surface+location.">Association of yeast transporters with detergent-resistant membranes correlates with their cell-surface location.</a> Traffic. 7, 1-15 (2006).</li><li>Kierszniowska, S., Seiwert, B., Schulze, W. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Definition+of+Arabidopsis+sterol-rich+membrane+microdomains+by+differential+treatment+with+methyl-&#223;-cyclodextrin+and+quantitative+proteomics.">Definition of Arabidopsis sterol-rich membrane microdomains by differential treatment with methyl-&#223;-cyclodextrin and quantitative proteomics.</a> Mol. Cell. Proteomics. 8, 612-623 (2009).</li><li>Simons, K., Toomre, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+rafts+and+signal+transduction.">Lipid rafts and signal transduction.</a> Nat. Rev. Mol. Cell Biol. 1, 31-39 (2000).</li><li>Maeda, Y., Kinoshita, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+remodeling,+trafficking+and+functions+of+glycosylphosphatidylinositol-anchored+proteins.">Structural remodeling, trafficking and functions of glycosylphosphatidylinositol-anchored proteins.</a> Prog. Lipid Res. 50, (2011).</li><li>Keinath, N. F., 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=PAMP-induced+changes+in+plasma+membrane+compartmentalization+reveal+novel+components+of+plant+immunity.">PAMP-induced changes in plasma membrane compartmentalization reveal novel components of plant immunity.</a> J. Biol. Chem. 285, 39140-39149 (2010).</li><li>Stanislas, T., 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=Quantitative+proteomics+reveals+a+dynamic+association+of+proteins+to+detergent+resistant+membranes+upon+elicitor+signaling+in+tobacco.">Quantitative proteomics reveals a dynamic association of proteins to detergent resistant membranes upon elicitor signaling in tobacco.</a> Mol. Cell. Proteomics. 8, 2186-2198 (2009).</li><li>Minami, 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=Alterations+in+detergent-resistant+plasma+membrane+microdomains+in+Arabidopsis+thaliana+during+cold+acclimation.">Alterations in detergent-resistant plasma membrane microdomains in Arabidopsis thaliana during cold acclimation.</a> Plant Cell Physiol. 50, 341-359 (2008).</li><li>Pauly, M., Eberhard, S., Albersheim, P., Darvill, A., York, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+the+mur1+mutation+on+xyloglucans+produced+by+suspension-cultured+Arabidopsis+thaliana+cells.">Effects of the mur1 mutation on xyloglucans produced by suspension-cultured Arabidopsis thaliana cells.</a> Planta. 214, 67-74 (2001).</li><li>Jouanneau, J. P., Peaud-Lenoel, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+and+synthesis+of+proteins+in+cell+suspensions+of+a+kinetin+dependent+tobacco.">Growth and synthesis of proteins in cell suspensions of a kinetin dependent tobacco.</a> Physiol. Plant. 20, 834-850 (1967).</li><li>Engelsberger, W. R., Erban, A., Kopka, J., Schulze, W. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+labeling+of+plant+cell+cultures+with+K15NO3+as+a+tool+for+quantitative+analysis+of+proteins+and+metabolites.">Metabolic labeling of plant cell cultures with K15NO3 as a tool for quantitative analysis of proteins and metabolites.</a> Plant Methods. 2, 1-11 (2006).</li><li>Arsova, B., Kierszniowska, S., Schulze, W. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+heavy+nitrogen+in+quantitative+proteomics+experiments+in+plants.">The use of heavy nitrogen in quantitative proteomics experiments in plants.</a> Trends Plant Sci. 17, 102-112 (2012).</li><li>Rappsilber, J., Ishihama, Y., Mann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stop+And+Go+Extraction+tips+for+matrix-assisted+laser+desorption/ionization,+nanoelectrospray,+and+LC/MS+sample+pretreatment+in+proteomics.">Stop And Go Extraction tips for matrix-assisted laser desorption/ionization, nanoelectrospray, and LC/MS sample pretreatment in proteomics.</a> Anal. Chem. 75, 663-670 (2003).</li><li>Olsen, J. V., Macek, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+accuracy+mass+spectrometry+in+large-scale+analysis+of+protein+phosphorylation.">High accuracy mass spectrometry in large-scale analysis of protein phosphorylation.</a> Methods Mol. Biol. 492, 131-142 (2009).</li><li>Schroeder, M. J., Shabanowitz, J., Schwartz, J. C., Hunt, D. F., Coon, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+neutral+loss+activation+method+for+improved+phosphopeptide+sequence+analysis+by+quadrupole+ion+trap+mass+spectrometry.">A neutral loss activation method for improved phosphopeptide sequence analysis by quadrupole ion trap mass spectrometry.</a> Anal. Chem. 76, 3590-3598 (2004).</li><li>Frese, C. K., 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=Improved+peptide+identification+by+targeted+fragmentation+using+CID,+HCD+and+ETD+on+an+LTQ-Orbitrap+Velos.">Improved peptide identification by targeted fragmentation using CID, HCD and ETD on an LTQ-Orbitrap Velos.</a> Journal of Protrome Research. 10, 2377-2388 (2011).</li><li>van Breukelen, B., vanden Toorn, H. W., Drugan, M. M., Heck, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=StatQuant:+a+post-quantification+analysis+toolbox+for+improving+quantitative+mass+spectrometry.">StatQuant: a post-quantification analysis toolbox for improving quantitative mass spectrometry.</a> Bioinformatics. 25, 1472-1473 (2009).</li><li>Zauber, H., Schulze, W. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomics+wants+cRacker:+Automated+standardized+data+analysis+of+LC/MS+derived+proteomic+data.">Proteomics wants cRacker: Automated standardized data analysis of LC/MS derived proteomic data.</a> J. Proteome Res. 11, 5548-5555 (2012).</li><li>Raffaele, S., Mongrand, S., Gamas, P., Niebel, A., Ott, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+annotation+of+remorins,+a+plant-specific+protein+family:+Evolutionary+and+functional+perspectives.">Genome-wide annotation of remorins, a plant-specific protein family: Evolutionary and functional perspectives.</a> Plant Physiol. 145, 593-600 (2007).</li><li>Shah, M. B., Sehgal, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nondetergent+isolation+of+rafts.">Nondetergent isolation of rafts.</a> Methods Mol. Biol. 398, 21-28 (2007).</li><li>Persaud-Sawin, D. -. A., Lightcap, S., Harry, G. J. <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+rafts+from+mouse+brain+tissue+by+a+detergent-free+method.">Isolation of rafts from mouse brain tissue by a detergent-free method.</a> Journal of Lipid Research. 50, 759-767 (2009).</li><li>Macdonald, L. J., Pike, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simplified+method+for+the+preparation+of+detergent-free+lipid+rafts.">A simplified method for the preparation of detergent-free lipid rafts.</a> Journal of Lipid Research. 46, 1061-1067 (2005).</li><li>M&#252;hlhaus, T., Weiss, J., Hemme, D., Sommer, F., Schroda, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+shotgun+proteomics+using+a+uniform+15N-labeled+standard+to+monitor+proteome+dynamics+in+time+course+experiments+reveals+new+insights+into+the+heat+stress+response+of+Chlamydomonas+reinhardtii.">Quantitative shotgun proteomics using a uniform 15N-labeled standard to monitor proteome dynamics in time course experiments reveals new insights into the heat stress response of Chlamydomonas reinhardtii.</a> Mol. Cell. Proteomics. 10, M110.004739 (2011).</li><li>Schulze, W., Usadel, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitation+in+mass-spectrometry-based+proteomics.">Quantitation in mass-spectrometry-based proteomics.</a> Annu. Rev. Plant Biol. 61, 491-516 (2010).</li><li>Foster, L. J., de Hoog, C., Mann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unbiased+quantiative+proteomics+of+lipid+rafts+reveales+high+specificity+for+signaling+factors.">Unbiased quantiative proteomics of lipid rafts reveales high specificity for signaling factors.</a> Proc. Natl. Acad. Sci. USA. 100, 5813-5818 (2003).</li><li>Lilley, K. S., Dupree, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+organelle+proteomics.">Plant organelle proteomics.</a> Curr. Opin. Plant Biol. 10, 594-599 (2007).</li><li>Sadowski, P. G., Groen, A. J., Dupree, P., Lilley, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sub-cellular+localization+of+membrane+proteins.">Sub-cellular localization of membrane proteins.</a> Proteomics. 8, 3991-4011 (2008).</li></ol>

The protocol presented in this paper contains many steps and all of them are crucial to obtain pure and representative fractionation of the plant plasma membrane into detergent resistant membranes and detergent soluble fractions. Therefore, it is important to follow each step as instructed.
Treatment of the plasma membrane fraction with non-ionic detergent (step 3.2) has the strongest influence on the quality of membrane microdomain fractionation. To obtain reproducible results between differe...

In 1972, Jonathan Singer and Garth Nicolson proposed the fluid mosaic model a structure model of cellular membranes, replacing the protein-lipid-protein sandwich model that was generally accepted in the early 1960s. Singer and Nicolson postulated that the biological membrane can be considered as a two-dimensional liquid where all lipid and protein molecules diffuse freely and easily 5. Since that time, structure model of the plasma membrane and knowledge of the membrane composition became even more complex. Particularly, within the plasma membrane, structures such as protein complexes and lipid/sterol based structurally disordered microdomains can be observed. In artificial model membranes 6,7, sterols and sphingolipids can laterally segregate from other lipid species to form regions with altered physical features. This segregation within the cellular membrane is mainly caused by the self-associating properties between sterols and highly saturated hydrocarbon chains of phopsho- and sphingolipids 8. Particularly, the rigid sterol rings favor interactions with stiffer and straighter saturated lipid species and these interactions force neighboring hydrocarbon chains into more extended conformations, increasing membrane thickness and hardness.
One of the commonly observed features of sterol enriched membrane microdomains was their insolubility upon treatment with non-ionic detergents such a Triton X-100 or Brij 35. These fractions were thought to be identical with membrane microdomains and were called detergent resistant membranes (DRM) based on their biochemical preparation method 2. The use of nonionic detergents during DRM extraction received some criticism as the biochemical DRM preparation may not directly correspond to any specific membrane compartment within the living cell 9. Particularly, the detergent to protein ratio seems crucial in such preparations, as different detergents, as well as different detergent amounts can yield different composition of the detergent resistant membrane fraction 10. However, there is evidence that particular protein species specifically associate with these cellular sterol-rich membrane domains, and that these proteins are well enriched in biochemical preparations of detergent-resistant membrane fractions 11. The core of proteins that were found in plant DRM fraction, and for which the presence in DRMs was sterol dependent, were particularly GPI-anchored proteins, such as fasciclin-like arabinogalactan proteins (FLAs) and members of the SKU protein family. Also some signaling proteins, such as receptor-like kinases or phospholipases were found 11. These results are consistent with many proteomic studies on mammalian membrane microdomains 12,13. Also in plants there is increasing evidence for the role of membrane microdomains in context of stress response 14-16.
The protocol described here provides a robust method for fractionation of plasma membrane microdomains and particularly uses a protein to detergent concentration that allows us to depict stress induced alterations of the sterol-rich membrane compartment 4,11,14.

<table border="1">
	<tbody>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>REAGENTS </td>
		</tr>
		<tr>
			<td>Chemicals were ordered from Sigma-Aldrich unless noted otherwise</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ammonia (stock 25 % solution)</td>
			<td>WAKO</td>
			<td>010-03166</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>TiO2 10 &mu;m</td>
			<td>GL-Science</td>
			<td>5020-75010</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Empore Disk C18</td>
			<td>Varian</td>
			<td>12145004</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Polyethylene glycol(PEG 3350)</td>
			<td>Sigma</td>
			<td>88276</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dextran T500</td>
			<td>Roth</td>
			<td>9219.2</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Promega</td>
			<td>V5113</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Protease inhibitor cocktail (PIC)</td>
			<td>Sigma</td>
			<td>P9599</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>K15NO3</td>
			<td>Cambridge Isotope Laboratories</td>
			<td>NLM-765-PK</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>EQUIPMENT </td>
		</tr>
		<tr>
			<td>Optima L-80 XP Ultracentrifuge</td>
			<td>Beckman</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Plate reader</td>
			<td>BioTek</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>EASY-nLC II nano-Liquid Chromatograph</td>
			<td>Thermo Scientific</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>LTQ-Orbitrap mass spectrometer</td>
			<td>Thermo Scientific</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Centrifuge 5810R</td>
			<td>Eppendorf</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Centrifuge 5417R</td>
			<td>Eppendorf</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Thermomixer</td>
			<td>Eppendorf</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Speed Vac RVC 2-25</td>
			<td>Christ</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Shaker Unimax 2010</td>
			<td>Heidolph</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

metabolic labeling and membrane fractionation for comparative proteomic analysis of arabidopsis thaliana suspension cell cultures

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling processes. Extracting sterol-enriched membrane microdomains from plant cells for proteomic analysis is a difficult task mainly due to multiple preparation steps and sources for contaminations from other cellular compartments. The plasma membrane constitutes only about 5-20% of all the membranes in a plant cell, and therefore isolation of highly purified plasma membrane fraction is challenging. A frequently used method involves aqueous two-phase partitioning in polyethylene glycol and dextran, which yields plasma membrane vesicles with a purity of 95% 1. Sterol-rich membrane microdomains within the plasma membrane are insoluble upon treatment with cold nonionic detergents at alkaline pH. This detergent-resistant membrane fraction can be separated from the bulk plasma membrane by ultracentrifugation in a sucrose gradient 2. Subsequently, proteins can be extracted from the low density band of the sucrose gradient by methanol/chloroform precipitation. Extracted protein will then be trypsin digested, desalted and finally analyzed by LC-MS/MS. Our extraction protocol for sterol-rich microdomains is optimized for the preparation of clean detergent-resistant membrane fractions from Arabidopsis thaliana cell cultures.
We use full metabolic labeling of Arabidopsis thaliana suspension cell cultures with K15NO3 as the only nitrogen source for quantitative comparative proteomic studies following biological treatment of interest 3. By mixing equal ratios of labeled and unlabeled cell cultures for joint protein extraction the influence of preparation steps on final quantitative result is kept at a minimum. Also loss of material during extraction will affect both control and treatment samples in the same way, and therefore the ratio of light and heave peptide will remain constant. In the proposed method either labeled or unlabeled cell culture undergoes a biological treatment, while the other serves as control 4.

With the presented protocol using metabolically labeled Arabidopsis cell cultures it is possible to isolate plasma membranes from plant tissue (step2, Figures 2 and 4), and enrich for detergent resistant membrane fractions within the plasma membrane (step 3, Figures 3 and 5). Subsequently, the protocol allows extraction of proteins from these detergent resistant membrane fractions (step 4) and digestion of the protein for comparative proteomic analysis (step 5). Finally, the opt...

Watch this Scientific Journal Video about Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures at JoVE.com

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Here we describe a robust method for the fractionation of plant plasma membranes into detergent resistant and detergent soluble membranes based on a mixture of unlabeled and in vivo fully 15N labeled Arabidopsis thaliana cell cultures. The procedure is applied for comparative proteomic studies to understand signaling processes.

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling ...

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Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::&beta;-glucuronidase Reporter Gene Expression

Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::&#946;-glucuronidase Reporter Gene Expression

The ability to introduce foreign genes into an organism is the foundation for modern biology and biotechnology. In the model flowering plant Arabidopsis ...

Measuring Cell Cycle Progression Kinetics with Metabolic Labeling and Flow Cytometry

Precise control of the initiation and subsequent progression through the various phases of the cell cycle are of paramount importance in proliferating ...

Metabolic Labeling of Leucine Rich Repeat Kinases 1 and 2 with Radioactive Phosphate

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are paralogs which share a similar domain organization, including a serine-threonine kinase domain, ...

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

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 ...

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

Arabidopsis thaliana is a model organism commonly used to understand and manipulate various cellular processes in plants, and it has been used extensively ...

Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures

Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures

The small intestinal mucosa exhibits a repetitive architecture organized into two fundamental structures: villi, projecting into the intestinal lumen and ...

Mixed Primary Cultures of Murine Small Intestine Intended for the Study of Gut Hormone Secretion and Live Cell Imaging of Enteroendocrine Cells

The gut is the largest endocrine organ of the body, with hormone-secreting enteroendocrine cells located along the length of the gastrointestinal ...

Analysis of Arabidopsis thaliana Growth Behavior in Different Light Qualities

Analysis of Arabidopsis thaliana Growth Behavior in Different Light Qualities

Plant biologists often need to observe the growth behavior of their chosen species. To this end, the plants need constant environmental and stable light ...

Spatiotemporal Investigation of ERL1 Dynamics via Confocal Imaging

Image-Based Methods to Study Membrane Trafficking Events in Stomatal Lineage Cells

Author Spotlight: Image-Based Methods to Study Membrane Trafficking Events in Stomatal Lineage Cells  

In eukaryotic cells, membrane components, including proteins and lipids, are spatiotemporally transported to their destination within the endomembrane ...