We thank Jennifer Deke and Urszula Zarzenska for their scientific support. This work was financially supported by a Career Integration Grant (CIG, PCIG14-GA-2013-63 0734) by the European Commission within the 7th framework program, the grant LFF-GK06 &#39;DELIGRAH&#39; (Landesforschungsf&#246;rderung Hamburg), and the DFG grant (DFG KE 856_6-1) awarded to JK.

1. Sampling and preparation of phloem sap from B. napus plants 4,8,20
<ol>
	<li>For phloem sap sampling, use well-watered 8-week-old B. napus plants that show only initial flowering to prevent pollen contaminations.</li>
	<li>Use a hypodermic needle (&#216; 0.8 mm) and punctate the inflorescence stem several times. Repeat at several other inflorescence stems and on other plants to obtain enough phloem sap (Figure 2a). 
	NOTE: For complex analysis, it is recommended to start with at least 600 &#181;L of phloem sap. 4 to 5 plants will yield between 200 and 700 &#181;L of phloem sap.</li>
	<li>Wipe off the first drop from the injured sites with filter paper. These drops mainly contain highly contaminated material from surrounding injured tissue and need to be discarded.</li>
	<li>Collect the following drops by pipetting and store the collected exudate in a pre-chilled conical 1.5 mL reaction tube at -20 &#176;C using an ice-free cooling system.</li>
	<li>Continue collecting until no further drop formation is observable, but no longer than 1 h. This procedure can be repeated after two days without significant loss of collected phloem sap. 
	NOTE: The collected phloem sap can be used immediately or stored at -80 &#176;C for future analysis. For -80 &#176;C storage, shock-freeze the reaction tube in liquid nitrogen.</li>
	<li>Concentrate the phloem sample to approximately 1/6th of the initial volume using centrifugal concentrators with a molecular weight cut off (MWCO) of 10,000 Daltons (Da). Centrifuge the concentrated sample at 4 &#176;C and 20,000 x g for at least 15 min to remove dust particles. 
	NOTE: Phloem sap concentration does not lead to a significant loss of proteins.</li>
	<li>For subsequent complex analysis, proceed with step 3.1.</li>
</ol>
2. Phloem sap purity control using reverse transcriptase PCR (RT-PCR) 4,8,20
<ol>
	<li>Isolate total RNA from phloem sap using a standard RNA extraction method for liquid material (e.g. TRIzol or phenol-chloroform extraction followed by isopropanol precipitation from the aqueous phase and ethanol washing steps according to the manufacturer&#39;s protocol). 
	Caution: Phenol-chloroform is toxic. Work under a hood with appropriate personal protection equipment. The extracted RNA can be stored in ethanol at -80 &#176;C for up to six months.</li>
	<li>Remove contamination of DNA by digestion with DNase I (1 u/&#181;g) for 45 min at 37 &#176;C and inactivate the enzyme by adding EDTA to a final concentration of 5 mM and incubate the sample at 75 &#176;C for 10 min.</li>
	<li>To get pure RNA, purify and concentrate the sample by another purification step (e.g. using RNA Cleanup Kits, according to the manufacturer&#39;s instructions).</li>
	<li>Perform a reverse transcriptase PCR (RT-PCR) for synthesis of cDNA with a cDNA synthesis kit using 150 ng of pure RNA as described by the manufacturer&#39;s protocol. 
	NOTE: The cDNA can be stored at -20 &#176;C until further use.</li>
	<li>Use 5 &#181;L of cDNA as a template for a standard PCR reaction as specified by the manufacturer, to amplify compartment-specific transcripts and check by agarose gel electrophoresis. Confirm the purity of phloem samples by e.g. using primers for rubisco small subunit, thioredoxin h, and the pollen coat protein (Figure 1b, Table 1).</li>
</ol>
3. Blue native PAGE 4,25,26,31
<ol>
	<li>Use either self-poured native gradient gels as described elsewhere27 or commercial Bis-Tris gradient gels.</li>
	<li>Load 20 to 30 &#181;g of the concentrated protein sample per well onto the gel in at least triplicates.</li>
	<li>Run the gel at 4 &#176;C and 150 V using dark blue cathode buffer B and anode buffer (Table 2) until the sample passed 1/3rd of the gel (ca. 20 to 30 min, Figure 3a). 
	NOTE: For northern blotting, a single gel lane is sufficient, while for the analysis of protein composition by 3D PAGE and mass spectrometry it is recommended to combine the same band from up to ten gel lanes to concentrate less abundant proteins.</li>
	<li>Pause the run and exchange dark blue cathode buffer B with light blue cathode buffer B/10 (Table 2) and continue the run. Finish the gel run when the blue front starts to elute out of the gel (ca. 120 to 180 min, Figure 3a). 
	NOTE: The finished blue native gel can be stored at 4 &#176;C for at least one week. Prolonged storage can lead to diffuse bands and should be avoided. Dark blue buffer B can be reused.</li>
</ol>
4. OPTIONAL: Detection of RNA using blue native northern blotting 4
<ol>
	<li>Cut out distinct bands or use the whole gel lane from the Blue native gel and transfer them onto a nylon membrane by semi-dry blotting at 3.5 mA/cm2 for 60 min and mark the upper and lower gel border with a pencil on the membrane (Figure 4a).</li>
	<li>After blotting, wash the membrane with DEPC-treated water and dry between two filter papers.</li>
	<li>Perform UV-crosslinking of the blotted membrane with 120,000 &#181;J/cm2 (85 s if using the Crosslinker in Table of Materials).</li>
	<li>Pre-hybridize the membrane with 6 mL of hybridization buffer (commercial or self-made) for 60 min at 68 &#176;C in a hybridization oven (Figure 4a).</li>
	<li>Add an additional 1 mL of hybridization buffer containing 4 &#181;L of 3&#39;-biotinylated probes (100 nM).</li>
	<li>Incubate the membrane with the probe overnight, while cooling the oven from 68 &#176;C to 37 &#176;C.</li>
	<li>Wash the membrane with buffer containing 2x SSC and 0.1 % SDS, for 5 min at room temperature to remove probes bound unspecifically. 
	Caution: SDS can cause irritations. Wear gloves and lab coat when working with SDS and SDS containing solutions.</li>
	<li>Perform the detection of the 3&#39;-biotinylated probes on the membrane e.g. with a Streptavidin-HRP-conjugate and luminol as a horseradish peroxidase (HRP) substrate, resulting in a sensitive chemiluminescent reaction (Figure 4a).</li>
</ol>
5. Second dimension: Tris-Tricine-urea PAGE 4,28
<ol>
	<li>Excise single bands from the blue native gel. Combine bands from samples run in parallel lanes to achieve a further concentration of complex proteins (Figure 5). 
	NOTE: Excised bands can be stored in single reaction tubes until further use at 4 &#176;C.</li>
	<li>Pour a 12 % Tris-Tricine-urea gel (thickness of 1 mm, 6.8cm x 8.6 cm (width x length)). Prepare 10 mL of gel solution A for the separating gel (Table 2), pour between two glass plates and overlay with isopropanol. Remove the isopropanol after the polymerization is finished and transfer 4 mL of gel solution B (Table 2) for the stacking gel onto the gel and insert a 10 well comb. 
	Caution: Unpolymerized acrylamide can be neurotoxic and TEMED is harmful if inhaled. Always wear personal protection equipment and work under a hood.</li>
	<li>Transfer the excised gel bands into a 1.5 mL reaction tube and add 1 mL of 2x SDS sample buffer for equilibration of the gel pieces (Figure 5, Table 2).
	<ol>
		<li>Incubate the samples for 10 min at room temperature, prior to boiling in a heating block (95 &#176;C) or in a microwave and incubate the samples again at room temperature for a further 15 min.</li>
		<li>Stack several equilibrated gel pieces representing the same complex into one single gel pocket.</li>
		<li>Perform the electrophoresis using anode and cathode running buffers at 150 V for 45 to 60 min and excise the whole gel lane.</li>
	</ol>
	</li>
	<li>Incubate the cut lane for 20 min in acidic buffer (100 mM Tris, 100 mM acetic acid) and equilibrate in 125 mM Tris-HCl, pH 6.8 for another 20 min (Figure 5).</li>
</ol>
6. Third dimension: SDS-PAGE 4,32
<ol>
	<li>Pour a 15 % SDS separating gel according to Laemmli32 (thickness: 1.5 mm, 6.8cm x 8.6 cm (Width x Length)) (Table 2) and add a thin layer of a 4 % stacking gel above. 
	Caution: SDS, acrylamide and TEMED are toxic and harmful. Work under a hood and wear personal protection equipment.</li>
	<li>Transfer the equilibrated gel lane onto the SDS gel and cover the gel with 0.5 % (w/w) agarose in 1x SDS running buffer supplemented with a trace of bromophenol blue (Figure 5).</li>
	<li>Boil the SDS running buffer with agarose in the microwave prior loading onto the gel.</li>
	<li>For running a protein marker to estimate the molecular weights of the single components, insert a piece of filter paper on one end of the gel until the agarose solidified or use a special comb typically used for IPG gels.</li>
	<li>Run the gel at 150 V for 60 min and stain the gel with either colloidal Coomassie, Silver stain or any other staining method suitable for subsequent mass spectrometric analysis.</li>
	<li>Analyze the visible protein spots using mass spectrometric approaches like matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) or LC-MS/MS mass spectrometry. 
	NOTE: We recommend to use the colloidal Coomassie staining as already described33. In our hands, even less abundant proteins can be sufficiently stained and allows a mass spectrometric analysis with reasonable data quality.</li>
</ol>

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Bot. 55 (402), 1473-1481 (2004).</li><li>Walz, C., Giavalisco, P., Schad, M., Juenger, M., Klose, J., Kehr, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomics+of+curcurbit+phloem+exudate+reveals+a+network+of+defence+proteins.">Proteomics of curcurbit phloem exudate reveals a network of defence proteins.</a> Phytochemistry. 65 (12), 1795-1804 (2004).</li><li>Walz, C., Juenger, M., Schad, M., Kehr, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+the+presence+and+activity+of+a+complete+antioxidant+defence+system+in+mature+sieve+tubes.">Evidence for the presence and activity of a complete antioxidant defence system in mature sieve tubes.</a> Plant J. 31 (2), 189-197 (2002).</li><li>Kehr, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phloem+sap+proteins:+their+identities+and+potential+roles+in+the+interaction+between+plants+and+phloem-feeding+insects.">Phloem sap proteins: their identities and potential roles in the interaction between plants and phloem-feeding insects.</a> J Exp. Bot. 57 (4), 767-774 (2006).</li><li>Ham, B. -. K., Brandom, J. L., Xoconostle-C&#225;zares, B., Ringgold, V., Lough, T. J., Lucas, W. 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+polypyrimidine+tract+binding+protein,+pumpkin+RBP50,+forms+the+basis+of+a+phloem-mobile+ribonucleoprotein+complex.">A polypyrimidine tract binding protein, pumpkin RBP50, forms the basis of a phloem-mobile ribonucleoprotein complex.</a> Plant Cell. 21 (1), 197-215 (2009).</li><li>Schamel, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biotinylation+of+protein+complexes+may+lead+to+aggregation+as+well+as+to+loss+of+subunits+as+revealed+by+Blue+Native+PAGE.">Biotinylation of protein complexes may lead to aggregation as well as to loss of subunits as revealed by Blue Native PAGE.</a> J. Immunol. Methods. 252 (1-2), 171-174 (2001).</li><li>Zhang, S., Sun, L., Kragler, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+phloem-delivered+RNA+pool+contains+small+noncoding+RNAs+and+interferes+with+translation.">The phloem-delivered RNA pool contains small noncoding RNAs and interferes with translation.</a> Plant Physiol. 150 (1), 378-387 (2009).</li></ol>

The authors have nothing to disclose.

The phloem is a compartment of high interest, since it constitutes the major transport route for photoassimilates, and is also essential for long-distance signaling between different plant parts9,20,34,35. In addition to small molecules, macromolecules like proteins and RNAs have been implicated with phloem maintenance and signaling4,7,8. The analysis of phloem composition is restricted by the limited accessibility to phloem samples in most plant species. The insect stylet technique is widely applicable, but experimentally demanding and results in very small amounts of phloem samples11. One easy technique used for phloem sampling is EDTA-facilitated exudation12. EDTA chelates divalent ions like Calcium and thus prevents closing of sieve plates by P-proteins and callose. It is easy to use, but is prone to contamination by damaged cells and samples are diluted. Depleting divalent ions by EDTA could also lead to a breakdown of existing complexes. However, it allows sampling from a wide range of species including the model plant A. thaliana15. Spontaneous exudation of phloem sap only occurs in a small number of plant species. It is an invasive method that involves significant injury of plant tissues10. We recently established B. napus as a model species for studying long-distance transport4,8,20. Here, phloem sap can be sampled from small incisions, which reduces wounding effects and sources of contamination.
Using different sampling techniques, the proteome of phloem samples from different plant species has been investigated in recent years7,8,17,36,37,38,39. For these proteome studies, proteins were extracted and precipitated under denaturing conditions and therefore do not allow conclusions about protein:protein and protein:nucleic acid interactions present under native conditions. Co-immunoprecipitation (CoIP) and overlay assays indicated that a major ribonucleoprotein complex might exist in phloem sap from pumpkin40, but it was not demonstrated that any macromolecular complexes exist under native conditions within the phloem system.
Blue native PAGE, introduced by Sch&#228;gger et al.26, in combination with subsequent high-resolution gel electrophoresis steps enable the separation of the individual components of large protein complexes. Using 3D-PAGE analyses of native B. napus phloem exudate, we could recently demonstrate that several high-molecular-weight protein:protein and RNP complexes exist in phloem samples and are enzymatically active4. Alternative methods to isolate protein complexes as well as RNPs like size exclusion chromatography might exist, but fail in terms of resolution when compared to BN-PAGE. Furthermore, for a reasonable quality of complex separation, size exclusion column matrices have to be adapted according to the overall complex molecular weights being investigated. Analyzing complexes of different size will therefore demand different types of columns which is cost intensive and does not allow as high focusing power as a BN-PAGE.
Critical steps to analyze complexes from B. napus include the total amount of phloem sap used as starting material. At least 600 &#181;L of phloem sample are necessary and can be obtained from five well-watered plants. For 3D-PAGE and BN northern blotting it is necessary to start the initial BN gel with a sufficient amount of phloem samples. To achieve this, phloem samples are concentrated until 20 to 30 &#181;g of protein can be loaded into each well and at least triplicates of the same sample are run in parallel. Too low or too high concentrations reduce the detection of complexes (Figure 3). Phloem samples need be collected into reaction tubes on ice and should be stored frozen for later use to avoid protein degradation and turnover. Protease inhibitors have been found in all phloem proteomes analyzed, but also a fully active proteasome was described in the phloem of B. napus4. Furthermore, volume and composition of phloem samples depend on the sampling time-point and environmental conditions10. Therefore care must be taken to collect at the same time of the day under similar conditions. Stress treatments (e.g. drought) can reduce the amount of phloem that can be collected. To identify single proteins by mass spectrometry, it is mandatory to limit possible keratin contaminations by wearing gloves all the time. Keratin leads to additional signals during mass spec analysis and hampers protein identification. Running the BN-PAGE at a higher voltage or at ambient temperature can lead to heating of the gel that might result in disassembly of fragile complexes.
The protocol presented here is suitable for the analysis of protein:protein complexes. We also show that it can be used to detect specific RNAs contained in the complexes in parallel. To achieve this, we recently introduced a protocol for BN northern blotting4. RNAs are prone to degradation by RNAse contaminations. To limit such degradation DEPC-treated, water should be used.
Main limitations of the presented method include the estimation of the molecular weight of protein complexes separated by BN-PAGE, since migration behavior depends on size, shape and even on posttranslational modifications of the complexes31,41. Size estimation of RNP complexes is even more complicated due to the influence of nucleic acids on the migration behavior. It can also not be prevented that complexes of the same size co-migrate in one single band. This can lead to the erroneous prediction of complex compositions. Therefore verifying the observed interactions with complementary techniques, e.g. by functional assays4, might be necessary. Also proteins only weakly or transiently associated to a macromolecular complex might be lost in the BN buffer used. To avoid this, samples could be crosslinked, what can also lead to artifacts or can prevent protein identification, or the buffer conditions can be optimized.
In contrast to classical 2D-PAGE, the combination of urea- and SDS-PAGE allows the separation according to hydrophobicity and molecular weight instead of isoelectric point (pI) and molecular weight 28, and does not limit the separation to proteins of a specific pI range. Similar to classical 2D-PAGE, separated proteins are visible as single spots, but on a diagonal line 4,28 (Figure 5, Figure 6). More hydrophobic proteins appear in spots above this diagonal, whereas more hydrophilic proteins are found below the line28. The polyacrylamide concentrations used in this protocol will separate proteins between 25 to 80 kDa well. To allow a separation of smaller or larger proteins the polyacrylamide concentration can be varied or gradient gels can be used in the third dimension. The components of complex IV (Figure 3) separated by 3D-PAGE (Figure 6) were cut out, trypsin digested, and analyzed by mass spectrometry. This resulted in the identification of several tRNA ligases. The components of the other complexes have been published recently 4. Our 3D-PAGE enabled the separation of different ligases, namely aspartate, asparagine, threonine, lysine and glycine ligases, with similar molecular weights (Table 3). Using a methionine tRNA-specific probe, we were able to detect potentially bound tRNA within complex IV, but if full-length tRNA or tRNA fragments are in the complex cannot be discriminated with the probes used. To check if the nucleic acids are really bound within the complex and not co-migrating, protease digested phloem sap samples might serve as suitable migration controls.
It has been speculated earlier that protein translation can occur within phloem sieve tubes, since almost most components of the entire ribosome as well as elongation factors have been found in phloem samples4,7. Our finding of tRNA and tRNA ligases seems to support this idea. However, tRNA fragments present in phloem samples from pumpkin have been shown to be potent inhibitors of translation42 and functional ribosomes seem to be absent4, suggesting that translation cannot take place.
Taken together, the protocol presented here allows the separation of native protein:protein and even RNP complexes from phloem samples and the separation and identification of their individual components with high resolution. The application of this method enables the discovery of novel protein and RNP complexes in phloem samples and can therefore elucidate new functions in this highly specialized plant compartment.

The long-distance conduits of the phloem are essential for the allocation of organic nutrients in higher plants, but are also involved in regulating many different processes important for growth and development, pathogen defense, and the adaptation to adverse environmental conditions. Besides small effectors like ions, phytohormones, or metabolites themselves, macromolecules like proteins and RNAs have recently emerged as potential signals.
Studies have shown that phloem loading of proteins is size dependent and controlled by the size exclusion limit (SEL) of the pore-plasmodesmal units connecting companion cells (CC) and sieve elements (SE) that are typically in the range of 10 to &#62;67 kDa1,2,3. However, despite these size limitations larger proteins and protein complexes have been found within the phloem system challenging the idea of size exclusion4, and even nonspecific loss of proteins from CC to SE has been suggested5.
Multiprotein as well as large ribonucleoprotein complexes play pivotal roles in biosynthesis and maintaining the structural integrity of the cell6. There is evidence that phloem-mobile protein-protein and ribonucleoprotein complexes exist4,7, but to date little about their function is known. In phloem sieve elements protein:protein and RNP complexes have been implicated with long-distance transport and/ or signaling8,9. To unravel the functions of the translocated complexes, studying their composition under varying environmental or stress conditions is required. To achieve this, native complexes have to be isolated and their components have to be separated with sufficient resolution.
To isolate high-molecular-weight complexes from the phloem, it is first necessary to obtain sap samples in sufficient quality and quantity. The technique that can be used for phloem sampling depends on the plant species of interest. Three main methods to obtain phloem sap exist 10: insect stylectomy11, EDTA-facilitated exudation12, and spontaneous exudation13.
Phloem samples from Arabidopsis thaliana (A. thaliana), the major model plant in laboratories worldwide, can only be obtained by EDTA-facilitated exudation or aphid stylectomy14,15. Both methods lead to either highly diluted phloem sap or very low sample amounts. EDTA-facilitated exudation is also prone to contamination and might not represent the real phloem composition16. Spontaneous phloem exudation is limited to plant species like cucurbits, lupine or yucca, where cutting off plant parts leads to a spontaneous emission of phloem sap that can be easily collected13,17,18,19. We recently established oilseed rape (Brassica napus) as a suitable model system for phloem analysis, since it is a close relative of A. thaliana and several microliters of phloem sap can be obtained from small incisions that has been shown to be of high purity4,8,20. Furthermore, the B. napus genome sequence was released in 201421.
Except for aphid stylectomy, all phloem collection methods described include damaging of surrounding tissue at the site of sampling, and the composition of phloem sap may change in response to injury. Therefore it is essential to check the quality of phloem samples, regardless of the sampling method applied. There are different methods for quality control of collected phloem sap, e.g. by determination of RNase activity22,23, RT-PCR with primers against Rubisco8,24, or the analysis of the sugar composition8.
Different methods to isolate and separate protein complexes can be applied, including size exclusion chromatography, sucrose density ultracentrifugation, or sample filtration through membranes with distinct molecular weight cut offs. However, these approaches do not allow high resolution. The technique called blue native PAGE (BN-PAGE), first described by Sch&#228;gger et al.25, is superior in terms of resolution. It was successfully used to determine the molecular weights of large native protein complexes from various organelles or from cellular lysates and their relative abundances26,27.
To further elucidate the complex composition of protein complexes, it is necessary to separate the individual components under denaturing conditions, leading to complete disassembly of the complex components. This can be achieved by a second dimension of SDS-PAGE 28,29 or, to achieve higher resolution, by a second dimension of isoelectric focusing (IEF) followed by a third dimension of SDS-PAGE30 and subsequent identification of the proteins using mass spectrometry.
In this protocol, we describe sampling of pure phloem sap from B. napus plants and the analysis of protein complexes from such phloem samples using a 3D electrophoretic approach combining BN-PAGE with Tris-Tricine-urea-PAGE and SDS-PAGE. The separated protein complex components are subsequently identified using mass spectrometry. In addition, we introduce blue native northern blotting as a method allowing the detection of RNAs in large RNP complexes4, extending the applicability of the approach.

<table>
	<tbody>
		<tr>
			<td>1.5 mL reaction tubes</td>
			<td>Eppendorf</td>
			<td>30120086</td>
			<td></td>
		</tr>
		<tr>
			<td>10 well comb for SDS PAGE, thickness 1 mm</td>
			<td>BioRad</td>
			<td>1653359</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Propanol</td>
			<td>AppliChem</td>
			<td>131090</td>
			<td></td>
		</tr>
		<tr>
			<td>30 % Acrylamide-bisacrylamide solution (29:1) solution</td>
			<td>BioRad</td>
			<td>1610156</td>
			<td></td>
		</tr>
		<tr>
			<td>30 % Acrylamide-bisacrylamide solution (37.5:1) solution</td>
			<td>BioRad</td>
			<td>1610158</td>
			<td></td>
		</tr>
		<tr>
			<td>96 % Ethanol</td>
			<td>Carl Roth</td>
			<td>P075</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic acid</td>
			<td>Carl Roth</td>
			<td>6755</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Lonza</td>
			<td>50004</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminum sulfate-(14-18)-hydrate</td>
			<td>AppliChem</td>
			<td>141101</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium peroxodisulfate (APS)</td>
			<td>Carl Roth</td>
			<td>9592.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Bis-Tris</td>
			<td>AppliChem</td>
			<td>A3992</td>
			<td></td>
		</tr>
		<tr>
			<td>Boric acid</td>
			<td>AppliChem</td>
			<td>A2940</td>
			<td></td>
		</tr>
		<tr>
			<td>Bromophenol blue</td>
			<td>AppliChem</td>
			<td>A2331</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifugal concentrators e.g. Vivaspin 500 (Mwco 10 kDa)</td>
			<td>Sartorius</td>
			<td>VS0102</td>
			<td></td>
		</tr>
		<tr>
			<td>Chemiluminescent Nucleic Acid Detection Module Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>89880</td>
			<td></td>
		</tr>
		<tr>
			<td>Chromatography papers e.g. 3 mm CHR</td>
			<td>Whatman</td>
			<td>3030-153</td>
			<td></td>
		</tr>
		<tr>
			<td>CoolBox M30 System</td>
			<td>Biocision</td>
			<td>BCS-133</td>
			<td></td>
		</tr>
		<tr>
			<td>Coomassie brilliant Blue G-250</td>
			<td>AppliChem</td>
			<td>A3480</td>
			<td></td>
		</tr>
		<tr>
			<td>Diethyl pyrocarbonate (DEPC)</td>
			<td>AppliChem</td>
			<td>A0881</td>
			<td></td>
		</tr>
		<tr>
			<td>Dithiothreitol (DTT)</td>
			<td>AppliChem</td>
			<td>A1101</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>AppliChem</td>
			<td>A3778</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol abs.</td>
			<td>AppliChem</td>
			<td>A3693</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid (EDTA)</td>
			<td>AppliChem</td>
			<td>A1103</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel Imaging system e.g. Chemidoc Touch</td>
			<td>BioRad</td>
			<td>1708370</td>
			<td></td>
		</tr>
		<tr>
			<td>GeneRuler 1kb plus</td>
			<td>Thermo Fisher Scientific</td>
			<td>SM1331</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>AppliChem</td>
			<td>141339</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>AppliChem</td>
			<td>131340</td>
			<td></td>
		</tr>
		<tr>
			<td>Heating block TS1</td>
			<td>Biometra</td>
			<td>846-051-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Hybridization oven e.g. GFL Hybridization Incubator 7601</td>
			<td>GFL</td>
			<td>7601</td>
			<td></td>
		</tr>
		<tr>
			<td>Hypodermic needle (0.8 mm)</td>
			<td>B Braun</td>
			<td>4658309</td>
			<td></td>
		</tr>
		<tr>
			<td>IPG gel comb, thickness 1 mm</td>
			<td>BioRad</td>
			<td>1653367</td>
			<td></td>
		</tr>
		<tr>
			<td>Labeling of RNA e.g.Biotin 3&#39;end DNA Labeling Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>89818</td>
			<td></td>
		</tr>
		<tr>
			<td>Native gradient gel e.g. Novex NativePAGE 4&#8211;16% Bis-Tris protein gel</td>
			<td>Invitrogen</td>
			<td>BN1002BOX</td>
			<td></td>
		</tr>
		<tr>
			<td>Nylon membrane e.g. Hybond-N+</td>
			<td>Amersham Pharmacia</td>
			<td>RPN203B</td>
			<td></td>
		</tr>
		<tr>
			<td>Ortho-phosphoric acid</td>
			<td>Carl Roth</td>
			<td>6366.1</td>
			<td></td>
		</tr>
		<tr>
			<td>PageRuler prestained protein ladder</td>
			<td>Thermo Fisher Scientific</td>
			<td>26616</td>
			<td></td>
		</tr>
		<tr>
			<td>Power supply for Gel electrophoresis EPS</td>
			<td>Amersham Pharmacia</td>
			<td>18-1130-01</td>
			<td>available from GE Healthcare</td>
		</tr>
		<tr>
			<td>RNA Cleanup Kit</td>
			<td>Qiagen</td>
			<td>74204</td>
			<td></td>
		</tr>
		<tr>
			<td>Semi dry blot e.g. Fastblot 43B semi dry Blot</td>
			<td>Biometra</td>
			<td>846-015-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>AppliChem</td>
			<td>A1149</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate (SDS)</td>
			<td>AppliChem</td>
			<td>142363</td>
			<td></td>
		</tr>
		<tr>
			<td>Synthesis of cDNA e.g. RevertAid First Strand cDNA Synthesis Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>K1621</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetramethylethylenediamine (TEMED)</td>
			<td>AppliChem</td>
			<td>A1148</td>
			<td></td>
		</tr>
		<tr>
			<td>Tricine</td>
			<td>AppliChem</td>
			<td>A3954</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>AppliChem</td>
			<td>A1086</td>
			<td></td>
		</tr>
		<tr>
			<td>Tri-sodium citrate dihydrate</td>
			<td>AppliChem</td>
			<td>A3901</td>
			<td></td>
		</tr>
		<tr>
			<td>TRIzol Reagent</td>
			<td>Thermo Fisher Scientific</td>
			<td>15596026</td>
			<td></td>
		</tr>
		<tr>
			<td>ULTRAhyb Ultrasensitive Hybridization Buffer</td>
			<td>Thermo Fisher Scientific</td>
			<td>AM8670</td>
			<td></td>
		</tr>
		<tr>
			<td>Urea</td>
			<td>AppliChem</td>
			<td>A1360</td>
			<td></td>
		</tr>
		<tr>
			<td>UV Crosslinker e.g. Stratalinker 2400</td>
			<td>Agilent</td>
			<td>NC0477671</td>
			<td>from Fisher Scientific</td>
		</tr>
		<tr>
			<td>Vertical electrophoresis apparatus e.g.The XCell SureLock Mini-Cell or Mini-Protean III System</td>
			<td>Thermo Fisher Scientific or BioRad</td>
			<td>EI0001 or 1658004</td>
			<td></td>
		</tr>
		<tr>
			<td>Wipers e.g. KIMWIPES Delicate Task Wipers</td>
			<td>Kimberly-Clark</td>
			<td>34120</td>
			<td></td>
		</tr>
	</tbody>
</table>

phloem sap sampling from brassica napus for 3d-page of protein and ribonucleoprotein complexes

Sampling the phloem of higher plants is often laborious and significantly dependent on the plant species. However, proteome studies under denaturing conditions could be achieved in different plant species. Native protein:protein and protein:nucleic acid complexes from phloem samples have as yet scarcely been analyzed, although they might play important roles in maintenance of this specialized compartment or in long-distance signaling. Large molecular assemblies can be isolated using a blue native gel electrophoresis (BN-PAGE). Their protein components can be separated by a subsequent sodium dodecyl sulfate PAGE (SDS-PAGE). However, proteins with similar molecular weights co-migrate, what can hinder protein identification by mass spectrometry. Combining BN-PAGE with two different denaturing gel electrophoresis steps, namely Tris-Tricine-urea and SDS-PAGE, enables the additional separation of proteins according to their hydrophilicity/hydrophobicity and thus increases resolution and the success of protein identification. It even allows distinguishing proteins that only differ in their posttranslational modifications. In addition, blue native northern blotting can be applied to identify the RNA components in macromolecular complexes. We show that our protocol is suitable to unravel the protein and RNA components of native protein:protein and ribonucleoprotein (RNP) complexes occurring in phloem samples. Combining a blue native PAGE with two different denaturing PAGE steps can help to separate different kinds of large protein complexes, and also enables an increased identification rate of their components by mass spectrometry. Furthermore, the protocol is robust enough to simultaneously detect potentially bound nucleic acids within single protein complexes.

Watch this Scientific Journal Video about Phloem Sap Sampling from Brassica napus for 3D-PAGE of Protein and Ribonucleoprotein Complexes at JoVE.com

Phloem Sap Sampling from Brassica napus for 3D-PAGE of Protein and Ribonucleoprotein Complexes | Biology | Video

Phloem Sap Sampling from Brassica napus for 3D-PAGE of Protein and Ribonucleoprotein Complexes

Here we present a protocol to analyze the protein composition of large native protein:protein and protein:nucleic acid complexes from oilseed rape (B. napus) phloem exudate using a 3D polyacrylamide gel electrophoresis (PAGE) approach combining blue native (BN) with two denaturing PAGEs followed by mass spectrometric identification.

Phloem Sap Sampling from Brassica napus for 3D-PAGE of Protein and Ribonucleoprotein Complexes

Sampling the phloem of higher plants is often laborious and significantly dependent on the plant species. However, proteome studies under denaturing ...

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13.7K Views.  Universität Hamburg. Here we present a protocol to analyze the protein composition of large native protein:protein and protein:nucleic acid complexes from oilseed rape (B. napus) phloem exudate using a 3D polyacrylamide gel electrophoresis (PAGE) approach combining blue native (BN) with two denaturing PAGEs followed by mass spectrometric identification.

Video: Phloem Sap Sampling from Brassica napus for 3D-PAGE of Protein and Ribonucleoprotein Complexes

Purification and Visualization of Influenza A Viral Ribonucleoprotein Complexes

The influenza A viral genome consists of eight negative-sense, single stranded RNA molecules, individually packed with multiple copies of the influenza A nucleoprotein (NP) into viral ribonulceoprotein particles (vRNPs). The influenza vRNPs are enclosed within the viral envelope. During cell entry, however, these vRNP complexes are released into the cytoplasm, where they gain access to the host nuclear transport machinery. In order to study the nuclear import of influenza vRNPs and the replication of the influenza genome, it is useful to work with isolated vRNPs so that other components of the virus do not interfere with these processes. Here, we describe a procedure to purify these vRNPs from the influenza A virus. The procedure starts with the disruption of the influenza A virion with detergents in order to release the vRNP complexes from the enveloped virion. The vRNPs are then separated from the other components of the influenza A virion on a 33-70% discontinuous glycerol gradient by velocity sedimentation. The fractions obtained from the glycerol gradient are then analyzed on via SDS-PAGE after staining with Coomassie blue. The peak fractions containing NP are then pooled together and concentrated by centrifugation. After concentration, the integrity of the vRNPs is verified by visualization of the vRNPs by transmission electron microscopy after negative staining. The glycerol gradient purification is a modification of that from Kemler et al. (1994)1, and the negative staining has been performed by Wu et al. (2007).2

The genome of the influenza A virus consists of eight separate complexes of RNA and proteins, termed viral ribonucleoprotein complexes (vRNPs). This paper describes the glycerol gradient purification and transmission electron microscopy visualization of influenza A vRNPs.

The influenza A viral genome consists of eight negative-sense, single stranded RNA molecules, individually packed with multiple copies of the influenza A ...

Identification of protein complexes with quantitative proteomics in S. cerevisiae

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as important intracellular signalling molecules. However, unlike proteins, lipids are small hydrophobic molecules that traffic primarily by poorly described nonvesicular routes, which are hypothesized to occur at membrane contact sites (MCSs). MCSs are regions where the endoplasmic reticulum (ER) makes direct physical contact with a partnering organelle, e.g., plasma membrane (PM). The ER portion of ER-PM MCSs is enriched in lipid-synthesizing enzymes, suggesting that lipid synthesis is directed to these sites and implying that MCSs are important for lipid traffic. Yeast is an ideal model to study ER-PM MCSs because of their abundance, with over 1000 contacts per cell, and their conserved nature in all eukaryotes. Uncovering the proteins that constitute MCSs is critical to understanding how lipids traffic is accomplished in cells, and how they act as signaling molecules. We have found that an ER called Scs2p localize to ER-PM MCSs and is important for their formation. We are focused on uncovering the molecular partners of Scs2p. Identification of protein complexes traditionally relies on first resolving purified protein samples by gel electrophoresis, followed by in-gel digestion of protein bands and analysis of peptides by mass spectrometry. This often limits the study to a small subset of proteins. Also, protein complexes are exposed to denaturing or non-physiological conditions during the procedure. To circumvent these problems, we have implemented a large-scale quantitative proteomics technique to extract unbiased and quantified data. We use stable isotope labeling with amino acids in cell culture (SILAC) to incorporate staple isotope nuclei in proteins in an untagged control strain. Equal volumes of tagged culture and untagged, SILAC-labeled culture are mixed together and lysed by grinding in liquid nitrogen. We then carry out an affinity purification procedure to pull down protein complexes. Finally, we precipitate the protein sample, which is ready for analysis by high-performance liquid chromatography/ tandem mass spectrometry. Most importantly, proteins in the control strain are labeled by the heavy isotope and will produce a mass/ charge shift that can be quantified against the unlabeled proteins in the bait strain. Therefore, contaminants, or unspecific binding can be easily eliminated. By using this approach, we have identified several novel proteins that localize to ER-PM MCSs. Here we present a detailed description of our approach. 

Here we describe a new quantitative proteomics technique for identifying protein complexes in Saccharomyces cerevisiae. In this study, we have used the SILAC method together with affinity purification followed by tandem mass spectrometry to identify with high specificity the binding partners of an ER protein, Scs2p.

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as ...

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

Analyzing Large Protein Complexes by Structural Mass Spectrometry

Living cells control and regulate their biological processes through the coordinated action of a large number of proteins that assemble themselves into an array of dynamic, multi-protein complexes1. To gain a mechanistic understanding of the various cellular processes, it is crucial to determine the structure of such protein complexes, and reveal how their structural organization dictates their function. Many aspects of multi-protein complexes are, however, difficult to characterize, due to their heterogeneous nature, asymmetric structure, and dynamics. Therefore, new approaches are required for the study of the tertiary levels of protein organization.
One of the emerging structural biology tools for analyzing macromolecular complexes is mass spectrometry (MS)2-5. This method yields information on the complex protein composition, subunit stoichiometry, and structural topology. The power of MS derives from its high sensitivity and, as a consequence, low sample requirement, which enables examination of protein complexes expressed at endogenous levels. Another advantage is the speed of analysis, which allows monitoring of reactions in real time. Moreover, the technique can simultaneously measure the characteristics of separate populations co-existing in a mixture. 
Here, we describe a detailed protocol for the application of structural MS to the analysis of large protein assemblies. The procedure begins with the preparation of gold-coated capillaries for nanoflow electrospray ionization (nESI). It then continues with sample preparation, emphasizing the buffer conditions which should be compatible with nESI on the one hand, and enable to maintain complexes intact on the other. We then explain, step-by-step, how to optimize the experimental conditions for high mass measurements and acquire MS and tandem MS spectra. Finally, we chart the data processing and analyses that follow. Rather than attempting to characterize every aspect of protein assemblies, this protocol introduces basic MS procedures, enabling the performance of MS and MS/MS experiments on non-covalent complexes. Overall, our goal is to provide researchers unacquainted with the field of structural MS, with knowledge of the principal experimental tools.

Mass spectrometry has proven to be a valuable tool for analyzing large protein complexes. This method enables insights into the composition, stoichiometry and overall architecture of multi-subunit assemblies. Here, we describe, step-by-step, how to perform a structural mass spectrometry analysis, and characterize macromolecular structures.

Living cells control and regulate their biological processes through the coordinated action of a large number of proteins that assemble themselves into an ...

A Noninvasive Hair Sampling Technique to Obtain High Quality DNA from Elusive Small Mammals

Noninvasive genetic sampling approaches are becoming increasingly important to study wildlife populations. A number of studies have reported using noninvasive sampling techniques to investigate population genetics and demography of wild populations1. This approach has proven to be especially useful when dealing with rare or elusive species2. While a number of these methods have been developed to sample hair, feces and other biological material from carnivores and medium-sized mammals, they have largely remained untested in elusive small mammals. In this video, we present a novel, inexpensive and noninvasive hair snare targeted at an elusive small mammal, the American pika (Ochotona princeps). We describe the general set-up of the hair snare, which consists of strips of packing tape arranged in a web-like fashion and placed along travelling routes in the pikas&#x2019; habitat. We illustrate the efficiency of the snare at collecting a large quantity of hair that can then be collected and brought back to the lab. We then demonstrate the use of the DNA IQ system (Promega) to isolate DNA and showcase the utility of this method to amplify commonly used molecular markers including nuclear microsatellites, amplified fragment length polymorphisms (AFLPs), mitochondrial sequences (800bp) as well as a molecular sexing marker. Overall, we demonstrate the utility of this novel noninvasive hair snare as a sampling technique for wildlife population biologists. We anticipate that this approach will be applicable to a variety of small mammals, opening up areas of investigation within natural populations, while minimizing impact to study organisms.

We present a noninvasive sampling approach to efficiently collect hair samples from elusive small mammals, as shown for the American pika. We demonstrate the utility of this method by extracting DNA from sampled hair and amplifying several types of molecular markers commonly used in studies of wildlife ecology and conservation.

Noninvasive genetic sampling approaches are becoming increasingly important to study wildlife populations. A number of studies have reported using ...

Biochemical Reconstitution of Steroid Receptor&#x2022;Hsp90 Protein  Complexes and Reactivation of Ligand Binding

Hsp90 is an essential and highly abundant molecular chaperone protein that has been found to regulate more than 150 eukaryotic signaling proteins, including transcription factors (e.g. nuclear receptors, p53) and protein kinases (e.g. Src, Raf, Akt kinase) involved in cell cycling, tumorigenesis, apoptosis, and multiple eukaryotic signaling pathways 1,2. Of these many 'client' proteins for hsp90, the assembly of steroid receptor&bull;hsp90 complexes is the best defined (Figure 1). We present here an adaptable glucocorticoid receptor (GR) immunoprecipitation assay and in vitro GR&bull;hsp90 reconstitution method that may be readily used to probe eukaryotic hsp90 functional activity, hsp90-mediated steroid receptor ligand binding, and molecular chaperone cofactor requirements. For example, this assay can be used to test hsp90 cofactor requirements and the effects of adding exogenous compounds to the reconstitution process. 
The GR has been a particularly useful system for studying hsp90 because the receptor must be bound to hsp90 to have an open ligand binding cleft that is accessible to steroid 3. Endogenous, unliganded GR is present in the cytoplasm of mammalian cells noncovalently bound to hsp90. As found in the endogenous GR&bull;hsp90 heterocomplex, the GR ligand binding cleft is open and capable of binding steroid. If hsp90 dissociates from the GR or if its function is inhibited, the receptor is unable to bind steroid and requires reconstitution of the GR&bull;hsp90 heterocomplex before steroid binding activity is restored 4 . GR can be immunoprecipitated from cell cytosol using a monoclonal antibody, and proteins such as hsp90 complexed to the GR can be assayed by western blot. Steroid binding activity of the immunoprecipitated GR can be determined by incubating the immunopellet with [3H]steroid. 
Previous experiments have shown hsp90-mediated opening of the GR ligand binding cleft requires hsp70, a second molecular chaperone also essential for eukaryotic cell viability. Biochemical activity of hsp90 and hsp70 are catalyzed by co-chaperone proteins Hop, hsp40, and p23 5. A multiprotein chaperone machinery containing hsp90, hsp70, Hop, and hsp40 are endogenously present in eukaryotic cell cytoplasm, and reticulocyte lysate provides a chaperone-rich protein source 6. 
In the method presented, GR is immunoadsorbed from cell cytosol and stripped of the endogenous hsp90/hsp70 chaperone machinery using mild salt conditions. The salt-stripped GR is then incubated with reticulocyte lysate, ATP, and K+, which results in the reconstitution of the GR&bull;hsp90 heterocomplex and reactivation of steroid binding activity 7. This method can be utilized to test the effects of various chaperone cofactors, novel proteins, and experimental hsp90 or GR inhibitors in order to determine their functional significance on hsp90-mediated steroid binding 8-11.

An in vitro method for preparing functional glucocorticoid receptor (GR)&#x2022;hsp90 protein complexes from purified proteins and cellular lysates is described. The method utilizes immunoadsorption of recombinant GR followed by salt-stripping and protein complex reconstitution. The importance of cofactors and buffer conditions are discussed, as are potential method applications.

Hsp90 is an essential and highly abundant molecular chaperone protein that has been found to regulate more than 150 eukaryotic signaling proteins, ...

Method for the Isolation and Identification of mRNAs, microRNAs and Protein Components of Ribonucleoprotein Complexes from Cell Extracts using RIP-Chip

As a result of the development of high-throughput sequencing and efficient microarray analysis, global gene expression analysis has become an easy and readily available form of data collection. In many research and disease models however, steady state levels of target gene mRNA does not always directly correlate with steady state protein levels. Post-transcriptional gene regulation is a likely explanation of the divergence between the two. Driven by the binding of RNA Binding Proteins (RBP), post-transcriptional regulation affects mRNA localization, stability and translation by forming a Ribonucleoprotein (RNP) complex with target mRNAs. Identifying these unknown de novo mRNA targets from cellular extracts in the RNP complex is pivotal to understanding mechanisms and functions of the RBP and their resulting effect on protein output. This protocol outlines a method termed RNP immunoprecipitation-microarray (RIP-Chip), which allows for the identification of specific mRNAs associated in the ribonucleoprotein complex, under changing experimental conditions, along with options to further optimize an experiment for the individual researcher. With this important experimental tool, researchers can explore the intricate mechanisms associated with post-transcriptional gene regulation as well as other ribonucleoprotein interactions.

A step by step protocol to isolating and identifying RNA associated complexes through RIP-Chip.

As a result of the development of high-throughput sequencing and efficient microarray analysis, global gene expression analysis has become an easy and ...

Identification of Post-translational Modifications of Plant Protein Complexes

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to infection via the recruitment and activation of immune complexes. Activation of immune complexes is associated with post-translational modifications (PTMs) of proteins, such as phosphorylation, glycosylation, or ubiquitination. Understanding how these PTMs are choreographed will lead to a better understanding of how resistance is achieved.
Here we describe a protein purification method for nucleotide-binding leucine-rich repeat (NB-LRR)-interacting proteins and the subsequent identification of their post-translational modifications (PTMs). With small modifications, the protocol can be applied for the purification of other plant protein complexes. The method is based on the expression of an epitope-tagged version of the protein of interest, which is subsequently partially purified by immunoprecipitation and subjected to mass spectrometry for identification of interacting proteins and PTMs.
This protocol demonstrates that: i). Dynamic changes in PTMs such as phosphorylation can be detected by mass spectrometry; ii). It is important to have sufficient quantities of the protein of interest, and this can compensate for the lack of purity of the immunoprecipitate; iii). In order to detect PTMs of a protein of interest, this protein has to be immunoprecipitated to get a sufficient quantity of protein.

We describe here a protocol for the purification and characterization of plant protein complexes. We demonstrate that by immunoprecipitating a single protein within a complex, so we can identify its post-translational modifications and its interacting partners.

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to ...

An in vivo Crosslinking Approach to Isolate Protein Complexes From Drosophila Embryos

Many cellular processes are controlled by multisubunit protein complexes. Frequently these complexes form transiently and require native environment to assemble. Therefore, to identify these functional protein complexes, it is important to stabilize them in vivo before cell lysis and subsequent purification. Here we describe a method used to isolate large bona fide protein complexes from Drosophila embryos. This method is based on embryo permeabilization and stabilization of the complexes inside the embryos by in vivo crosslinking using a low concentration of formaldehyde, which can easily cross the cell membrane. Subsequently, the protein complex of interest is immunopurified followed by gel purification and analyzed by mass spectrometry. We illustrate this method using purification of a Tudor protein complex, which is essential for germline development. Tudor is a large protein, which contains multiple Tudor domains - small modules that interact with methylated arginines or lysines of target proteins. This method can be adapted for isolation of native protein complexes from different organisms and tissues.

An in vivo Crosslinking Approach to Isolate Protein Complexes From Drosophila Embryos

Multi-component protein complexes play crucial roles during cellular function and development. Here we describe a method used to isolate native protein complexes from Drosophila embryos after in vivo crosslinking followed by purification of the crosslinked complexes for subsequent structure-function analysis.

Many cellular processes are controlled by multisubunit protein complexes. Frequently these complexes form transiently and require native environment to ...

Sampling Blood from the Lateral Tail Vein of the Rat

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, proteins, and glucose, yet the composition of the blood is dynamically regulated and easily perturbed. One factor that can change the blood composition is the stress response triggered by the sampling procedure, which can contribute to variability in the measures of interest. Here we describe a procedure for blood sampling from the lateral tail vein in the rat. This procedure offers significant advantages over other more commonly used techniques. It permits rapid sampling with minimal pain or invasiveness, without anesthesia or analgesia. Additionally, it can be used to obtain large volume samples (upwards of 1 ml in some rats), and it may be used repeatedly across experimental days. By minimizing the stress response and pain resulting from blood sampling, measures can more accurately reflect the true basal state of the animal, with minimal influence from the sampling procedure itself.

Blood samples are useful for assessing biomarkers of physiological states or disease in vivo. Here we describe the methodology to sample blood from the lateral tail vein in the rat. This method provides rapid samples with minimal pain and invasiveness.

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, ...