The work was supported by grants from the Swiss National Science Foundation (31003A_156998 and 31003A_176191) and by the University of Neuch&#226;tel.

1. Preparation of Arabidopsis Plants
<ol>
	<li>Prepare 1 L of half strength of Murashige and Skoog (MS) medium including vitamins with 1% sucrose and adjust the pH to 5.7 with potassium hydroxide (KOH). Add 0.8% of phytoagar and autoclave for 20 min at 120 &#176;C.</li>
	<li>Pour 75 mL of MS medium per Petri plate (diameter 14.5 cm, height 3 cm) and allow the solidification for 1 h.</li>
	<li>Add 30 mg of Arabidopsis thaliana seeds to a 1.5 mL microfuge tube, and surface sterilize with 70% (v/v) ethanol containing 0.05% (v/v) Triton X-100 for 5 min, followed by 100% (v/v) ethanol for 10 min. Remove the ethanol.</li>
	<li>Transfer the seeds to sterile filter paper for drying. Sprinkle the sterilized seeds evenly onto a MS plate (each genotype requires at least 8-10 plates), and seal each plate with surgical tape.</li>
	<li>Incubate the plates at 4 &#176;C for two days in the dark to synchronize seed germination. Transfer to a growth chamber with the following light cycle: 8 h of 120 &#181;mol m&#8722;2 s&#8722;1 light, and 16 h of dark at 22-20 &#176;C.</li>
</ol>
2. Preparation of HsIgG Agarose Beads
<ol>
	<li>Suspend CNBr-activated agarose beads (4 g) in 100 mL of 1 mM HCl and incubate for 30 min at room temperature.</li>
	<li>Transfer the agarose beads into a sintered glass filter, wash under vacuum with 100 mL of 100 mM HCl and repeat 2-3 times. 
	NOTE: Precool the wash solution to 4 &#176;C.</li>
	<li>Resuspend the agarose beads in 1 mL of cold 100 mM HCl and transfer to a 50 mL conical centrifuge tube. Wash the sintered glass filter with 3 mL of 100 mM HCl and transfer the liquid to the same tube.</li>
	<li>Spin at 400 &#215; g for 5 min at 4 &#176;C and remove the supernatant with a pipette. 
	NOTE: Do not decant the tube.</li>
	<li>Resuspend the beads in 50 mL of coupling buffer (Table 1); mix briefly, and then spin at 400 &#215; g for 5 min at 4 &#176;C.</li>
	<li>Dissolve 50 mg of lyophilized Homo sapiens IgG (Hs-IgG) in 10 mL of coupling buffer, mix the agarose beads gently, and incubate in rotary shaker overnight at 4 &#176;C.</li>
	<li>Spin at 400 &#215; g for 5 min at 4 &#176;C and remove the supernatant with a pipette.</li>
	<li>Wash the IgG-agarose beads with 50 mL of coupling buffer and spin at 400 &#215; g for 5 min at 4&#176;C.</li>
	<li>Resuspend the IgG-agarose beads with 50 mL of blocking buffer (Table 1) and spin at 400 &#215; g for 5 min at 4 &#176;C. Repeat once.</li>
	<li>Resuspend IgG-agarose beads in 50 mL of blocking buffer, rotate the mixture for 2 h at RT and spin at 400 &#215; g for 5 min at 4 &#176;C.</li>
	<li>Remove the supernatant and resuspend the IgG-agarose beads in 200 mL of NaCl coupling buffer (Table 1).</li>
	<li>To block any remaining cross-linking CNBr groups, wash the IgG-agarose beads with 100 mL of 0.1 M glycine-HCl, pH 2.8. Spin at 400 &#215; g for 5 min at 4 &#176;C and remove the supernatant. Wash with 0.2 M glycine-HCl, pH 2.8, spin at 400 &#215; g for 5 min at 4 &#176;C and remove the supernatant. Finally, wash the IgG-agarose beads with 200 mL of ultrapure water.</li>
	<li>Wash the IgG-agarose beads with 200 mL of ultrapure water and then 200 mL of PBS buffer (Table 1).</li>
	<li>Resuspend the IgG-agarose beads in 20 mL of PBS buffer with 0.01% NaN3 (sodium azide) and store at 4 &#176;C.</li>
</ol>
3. Isolation and Solubilization of the Membrane Fraction
<ol>
	<li>Grind 10 g of three weeks old A. thaliana seedlings in liquid nitrogen using a cold mortar and pestle with sand.</li>
	<li>Add 20 mL of cold grinding buffer (Table 1) to 10 g of ground tissue, mix well, and thaw on ice.</li>
	<li>Filter the homogenate through two layers of quick filtration material into a 50mL conical centrifuge tube. Soak the quick filtration material with grinding buffer before filtration.</li>
	<li>Centrifuge the filtrate for 10 min at 1,500 &#215; g at 4 &#176;C and transfer the supernatant to a chilled 50 mL conical centrifuge tube, repeat the same step one more time and collect the supernatant. Retain a 200 &#181;L sample of the &#34;total fraction&#34; and store at -80 &#176;C for later analysis</li>
	<li>Transfer the supernatant to cold 38.50 mL ultracentrifuge tubes and top up to 35 mL with cold grinding buffer. Centrifuge for 1 h at 100,000 &#215; g at 4 &#176;C in an ultracentrifuge.</li>
	<li>Transfer the supernatant to a 50 mL conical centrifuge tube. Retain a 200 &#181;L sample of the &#34;soluble fraction&#34; and store at -80 &#176;C for later analysis.</li>
	<li>Re-suspend the green pellet using a glass teflon homogenizer, in grinding buffer. Centrifuge for 1 h at 100,000 &#215; g at 4 &#176;C in an ultracentrifuge and discard the supernatant.</li>
	<li>Resuspend the green pellet in 18.75 mL of 1x grinding Buffer using a glass teflon homogenizer and add 9.375 mL of 1x grinding buffer.</li>
	<li>Add 9.375 mL of 4x solubilisation solution (Table 1) (containing the TX-100 detergent) to give 37.5 mL and incubate for 30 min on a &#34;rotating shaker&#34; at 4 &#176;C.</li>
	<li>Centrifuge for 1 h at 100,000 &#215; g at 4 &#176;C in an ultracentrifuge. Transfer the supernatant to a 50 mL conical centrifuge tube. Retain a 200 &#181;L sample of the supernatant (future &#34;load fraction&#34;) and store at -80 &#176;C for later analysis.</li>
	<li>Resuspend the pellet in 37.5 mL of grinding buffer. Retain the sample of insoluble part and store at -80 &#176;C</li>
</ol>
4. Immunoprecipitation
<ol>
	<li>Equilibrate 100 &#181;L of packed IgG-agarose resin in Buffer A (Table 1) and spin at 100 &#215; g for 5 min at 4 &#176;C.</li>
	<li>Remove the supernatant and resuspend the IgG-agarose resin in 100 &#181;L of Buffer A at 4 &#176;C.</li>
	<li>Transfer the washed IgG-agarose resin to the 37.5 mL of solubilized membranes and incubate overnight on a rotating shaker in a 50 mL conical centrifuge tube at 4 &#176;C.</li>
	<li>Sediment the IgG-agarose resin at 100 &#215; g for 5 min at 4 &#176;C and remove the supernatant with a pipette. Retain a 200 &#181;L sample of the &#34;flow through&#34; and store at -80 &#176;C for later analysis.</li>
	<li>Wash the IgG-agarose resin in 37.5 mL of Buffer A and incubate for 10 min on a rotating shaker.</li>
	<li>Sediment the IgG-agarose resin at 100 &#215; g for 5 min at 4 &#176;C, remove the supernatant with a pipette, add 5 mL of grinding Buffer A to a 15 mL centrifuge tube and centrifuge at 100 &#215; g, 4 &#176;C, 5 min. Retain a 200 &#181;L sample of the &#34;Wash 1&#34; and store at -80 &#176;C for later analysis.</li>
	<li>Repeat the 5 mL washing step three times with Buffer A.</li>
	<li>Carry out the last two washing steps without the inhibitors (NaF, Protease inhibitor and PMSF) 
	NOTE: Inhibitors are no longer required at this stage and omitted to reduce cost. Retain a 200 &#181;L sample of the last washing step &#34;final wash (W6)&#34; and store at -80 &#176;C for later analysis.</li>
	<li>Transfer the IgG-agarose resin to a 500 &#181;L spin column with a 35 &#181;m filter and wash the beads twice with 300 &#181;L of TEV elution buffer (Table 1) (not containing AcTEV) for equilibration. Close the spin column.</li>
	<li>Add 300 &#181;L of TEV elution buffer containing 50 units (10 &#181;L) of AcTEV. Prepare the mix in a tube before adding to the resin.</li>
	<li>Incubate the spin column with beads in a thermomixer at 16 &#176;C, 350 rpm for 2 h. Invert the column gently several times every 30 min.</li>
	<li>Open the spin column, place it in a new clean 1.5 mL tube and spin at 100 &#215; g for 5 min at 4 &#176;C to collect the eluate.</li>
	<li>Dry (for instance) 10% (v/v) of the eluted sample (~25 &#181;L) in a centrifugal evaporator and use for mass spectrometry or other further analysis.</li>
	<li>Aliquot the remaining eluate (~275 &#181;L) divided in to eleven 1.5 mL centrifuge tubes (25 &#181;L each), dry in a centrifugal evaporator, and store at -80 &#176;C.</li>
	<li>Analyze the samples retained throughout the procedure as well as the eluates by standard SDS-PAGE followed by immunoblotting. For sample preparation, precipitate 50 &#181;g of Total (T), Load (L), Flow through (FT), Wash 1 (W1) and 200 &#181;L of Wash 6 (W6) by chloroform/methanol extraction16.</li>
	<li>Add 10 &#181;L of 2x SDS-PAGE loading buffer (Table 1) to the precipitated samples and 25 &#181;L of eluted dried sample. Vortex and boil for 10 min at 95 &#176;C in a heat block.</li>
	<li>Analyze Total (T), Load (L), Flow through (FT), Wash 1 (W1), Wash 6 (W6) and Elution (E) by immunoblotting using anti-TOC159 antibodies to determine the efficiency of the isolation procedure.</li>
</ol>

<ol>
	<li>Jarvis, P., L&#243;pez-Juez, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biogenesis+and+homeostasis+of+chloroplasts+and+other+plastids.">Biogenesis and homeostasis of chloroplasts and other plastids.</a> Nature Reviews Molecular Cell Biology. 14 (12), 787-802 (2013).</li><li>Kessler, F., Schnell, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chloroplast+biogenesis:+diversity+and+regulation+of+the+protein+import+apparatus.">Chloroplast biogenesis: diversity and regulation of the protein import apparatus.</a> Current opinion in cell biology. 21 (4), 494-500 (2009).</li><li>Schnell, D. J., Kessler, F., Blobel, G. <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+components+of+the+chloroplast+protein+import+machinery.">Isolation of components of the chloroplast protein import machinery.</a> Science. 266 (5187), 1007-1012 (1994).</li><li>Kessler, F., Blobel, G., Patel, H. A., Schnell, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+two+GTP-binding+proteins+in+the+chloroplast+protein+import+machinery.">Identification of two GTP-binding proteins in the chloroplast protein import machinery.</a> Science. 266 (5187), 1035-1039 (1994).</li><li>Jarvis, P., 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=An+Arabidopsis+mutant+defective+in+the+plastid+general+protein+import+apparatus.">An Arabidopsis mutant defective in the plastid general protein import apparatus.</a> Science. 282 (5386), 100-103 (1998).</li><li>Bauer, J., 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=Essential+role+of+the+G-domain+in+targeting+of+the+protein+import+receptor+atToc159+to+the+chloroplast+outer+membrane.">Essential role of the G-domain in targeting of the protein import receptor atToc159 to the chloroplast outer membrane.</a> The Journal of cell biology. 159 (5), 845-854 (2002).</li><li>Kovacs-Bogdan, E., Soll, J., Bolter, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+import+into+chloroplasts:+the+Tic+complex+and+its+regulation.">Protein import into chloroplasts: the Tic complex and its regulation.</a> Biochimica et biophysica acta. 1803 (6), 740-747 (2010).</li><li>Nakai, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+TIC+complex+uncovered:+The+alternative+view+on+the+molecular+mechanism+of+protein+translocation+across+the+inner+envelope+membrane+of+chloroplasts.">The TIC complex uncovered: The alternative view on the molecular mechanism of protein translocation across the inner envelope membrane of chloroplasts.</a> Biochimica et biophysica acta. 1847 (9), 957-967 (2015).</li><li>Kikuchi, S., 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=Uncovering+the+protein+translocon+at+the+chloroplast+inner+envelope+membrane.">Uncovering the protein translocon at the chloroplast inner envelope membrane.</a> Science. 339 (6119), 571-574 (2013).</li><li>Kohler, D., 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=Characterization+of+chloroplast+protein+import+without+Tic56,+a+component+of+the+1-megadalton+translocon+at+the+inner+envelope+membrane+of+chloroplasts.">Characterization of chloroplast protein import without Tic56, a component of the 1-megadalton translocon at the inner envelope membrane of chloroplasts.</a> Plant physiology. 167 (3), 972-990 (2015).</li><li>Zufferey, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+novel+chloroplast+outer+membrane+kinase+KOC1+is+a+required+component+of+the+plastid+protein+import+machinery.">The novel chloroplast outer membrane kinase KOC1 is a required component of the plastid protein import machinery.</a> The Journal of biological chemistry. 292 (17), 6952-6964 (2017).</li><li>Rigaut, G., 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=A+generic+protein+purification+method+for+protein+complex+characterization+and+proteome+exploration.">A generic protein purification method for protein complex characterization and proteome exploration.</a> Nature biotechnology. 17 (10), 1030-1032 (1999).</li><li>Rohila, J. S., Chen, M., Cerny, R., Fromm, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+tandem+affinity+purification+tag+and+methods+for+isolation+of+protein+heterocomplexes+from+plants.">Improved tandem affinity purification tag and methods for isolation of protein heterocomplexes from plants.</a> The Plant journal. 38 (1), 172-181 (2004).</li><li>Rohila, J. S., 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=Protein-protein+interactions+of+tandem+affinity+purified+protein+kinases+from+rice.">Protein-protein interactions of tandem affinity purified protein kinases from rice.</a> PloS one. 4 (8), 6685 (2009).</li><li>Agne, B., 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+acidic+A-domain+of+Arabidopsis+TOC159+occurs+as+a+hyperphosphorylated+protein.">The acidic A-domain of Arabidopsis TOC159 occurs as a hyperphosphorylated protein.</a> Plant physiology. 153 (3), 1016-1030 (2010).</li><li>Wessel, D., Flugge, U. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+the+quantitative+recovery+of+protein+in+dilute+solution+in+the+presence+of+detergents+and+lipids.">A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids.</a> Analytical biochemistry. 138 (1), 141-143 (1984).</li></ol>

The authors declare no competing interests.

We demonstrated that a single step TAP tag purification is a specific and efficient method for the purification of the Arabidopsis TOC-TIC complex. Although the TAP-tag would allow two successive purification steps (IgG- followed by calmodulin affinity purification after TEV protease-cleavage), we believe that in many cases the first affinity step followed by TEV protease cleavage will be sufficient. Our target, TOC159, is low abundant and the inclusion of the second calmodulin-affinity step excessively diminished the protein yield which was the main reason for excluding it from our present protocol. The TEV-elution in itself is a highly specific and gentle purification step that will only release the TAP-tagged target together with associated interaction partners while contaminating the proteins sticking non-specifically to the IgG resin will remain there. Thus, in combination with the IgG-affinity step, the TEV elution results in a sufficiently efficient purification for our and probably many others&#39; purposes.
Care must be taken that the TAP-tagged construct is fully functional in vivo. TAP-tagging could have potentially deleterious effects on protein activity, stability or localization. TOC159 consists of three domains, the N-terminal acidic domain, the central GTP-binding domain and C-terminal membrane domain, that anchors TOC159 in the outer chloroplast membrane2. To avoid potential interference with membrane insertion, we fused the TAP-tag to the N-terminus of TOC159. Fusion to the N-terminus is rather the exception than the rule. Many proteins contain N-terminal targeting information and should therefore by tagged at the C-terminus. We assured that TOC159 is functional in vivo by complementation of the ppi2 mutant (an albino mutant lacking TOC159) with TAP-TOC159. The rescue of the green, wild type phenotype indicated that TAP-TOC159 was functional15.
The purification protocol was used to identify new interaction partners of TOC159, that included KOC1 and TIC5610,11. In total, around 30 proteins were associated with the complex suggesting that new interaction partners will be isolated and characterized in the near future. While we highly recommend the TAP-tag for complex purification, we would still like to point out that additional versions to the one presented here exist and may be very useful for some applications.

Plants depend on chloroplasts for photosynthesis and photoautotrophic growth1. The vast majority of chloroplast proteins are encoded in the nucleus, synthesized in the cytosol and imported into the chloroplast via the TIC and TOC complexes in the envelope membranes2. The core of the TOC complex consists of the Toc75 protein-conducting channel and the two receptor GTPases Toc33 and Toc1593,4,5. TOC159 is essential for the biogenesis of chloroplasts and mediates the massive accumulation of photosynthesis-associated proteins6. The TIC complex consists of the TIC20 protein-conducting channel as well as TIC110 and TIC407,8. Recently, a 1MD protein translocation complex at the inner envelope membrane was isolated and contained previously unidentified components9, one of which was TIC56. We recently co-isolated TIC56 of the 1 MDa complex using the TAP-TOC159 affinity purification protocol. The data indicated a structural overlap between the &#34;canonical&#34; TOC/TIC complexes and the 1 MDa complex10. Previously unknown components such as KOC1 were also identified as new interaction partners of the TOC and TIC complexes using the TAP-method described here10,11.
Thus, the TAP-tag purification is an efficient method for the isolation of protein complexes and the identification of interacting partners by subsequent mass spectrometric analysis12. The TAP tag consists of two IgG binding repeats separated from a calmodulin-binding peptide (CBP) by a tobacco etch virus (TEV) protease cleavage site. The original method, consisting of an IgG-affinity purification step followed by TEV cleavage and subsequent calmodulin-affinity chromatography permitted the native purification of large and highly pure protein complexes13,14. We have simplified the procedure and demonstrated that the TOC159-containing protein complexes can also be purified efficiently using only the IgG-affinity purification step followed by TEV-cleavage for elution15. In our experience, the omission of the calmodulin-affinity step resulted in higher yields and may therefore be appropriate for low abundance proteins.
In short, we engineered stable transgenic A. thaliana lines expressing TAP-TOC159 in the ppi2 (toc159 KO mutant) background and established it as a reliable source for the purification of TOC-TIC complexes. The protocol for the isolation of the TOC-TIC complex starts with the homogenization of the plant material in a detergent-free buffer. After centrifugation, the supernatant is discarded. The pellet containing the total membrane fraction is solubilized using a detergent-containing buffer. After an ultra-centrifugation step, the supernatant containing solubilized TOC-TIC complexes is applied to the IgG-resin for affinity purification. After several washing steps, elution is carried out using a TEV protease-containing buffer to selectively cleave downstream of the IgG-binding domains and gently release native TOC159-containing complexes. The TEV eluate can be analyzed directly by Western blotting or mass spectrometry to identify the interaction partners of TOC15910,11,15. The method has also been used to identify post-translational modifications of TOC15915. In the future, native TOC159-containing complexes may be used for structural studies using cryoelectron microscopy.

<table border="0" cellpadding="0" cellspacing="0" style="width:783px;" width="783">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="25">
			<td height="25" style="height:25px;width:312px;">NaHCO3</td>
			<td style="width:185px;">PanReac&#160; AppliChem</td>
			<td style="width:119px;">A0384</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Tris</td>
			<td style="width:185px;">Biosolve</td>
			<td style="width:119px;">0020092391BS</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:312px;">Na2HPO4</td>
			<td style="width:185px;">Sigma</td>
			<td style="width:119px;">S7909</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:312px;">KH2PO4</td>
			<td style="width:185px;">PanReac&#160; AppliChem</td>
			<td style="width:119px;">A2946</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">NaCl</td>
			<td style="width:185px;">PanReac&#160; AppliChem</td>
			<td style="width:119px;">A2942</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">NaF</td>
			<td style="width:185px;">Sigma</td>
			<td style="width:119px;">S1509</td>
			<td>Toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">PMSF</td>
			<td style="width:185px;">PanReac&#160; AppliChem</td>
			<td style="width:119px;">A0999</td>
			<td>Toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Plant protease inhibitor cocktail</td>
			<td style="width:185px;">Sigma</td>
			<td style="width:119px;">P9599</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Glycerol</td>
			<td style="width:185px;">PanReac&#160; AppliChem</td>
			<td style="width:119px;">A0970</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Triton X100</td>
			<td style="width:185px;">Roche</td>
			<td style="width:119px;">10789704001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Glycine</td>
			<td style="width:185px;">PanReac&#160; AppliChem</td>
			<td style="width:119px;">A1067</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">EDTA</td>
			<td style="width:185px;">Carl Roth</td>
			<td style="width:119px;">8043</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Dithiothreitol (DTT)</td>
			<td style="width:185px;">PanReac&#160; AppliChem</td>
			<td style="width:119px;">A1101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">SDS</td>
			<td style="width:185px;">PanReac&#160; AppliChem</td>
			<td style="width:119px;">A0675</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Bromophenol blue</td>
			<td style="width:185px;">Sigma</td>
			<td style="width:119px;">B0126</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">HCl</td>
			<td style="width:185px;">VWR</td>
			<td style="width:119px;">20252-290</td>
			<td>Corrosive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Sucrose</td>
			<td style="width:185px;">PanReac&#160; AppliChem</td>
			<td style="width:119px;">A3935</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:312px;">Murashige and Skoog medium with vitamins</td>
			<td style="width:185px;">Duchefa Biochemie</td>
			<td style="width:119px;">M0222</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Phytoagar</td>
			<td style="width:185px;">Duchefa Biochemie</td>
			<td style="width:119px;">P1003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Petri plate</td>
			<td style="width:185px;">Greiner Bio-one</td>
			<td style="width:119px;">639102</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">1.5 mL microfuge tubes</td>
			<td style="width:185px;">Sarstedt</td>
			<td style="width:119px;">72.706</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Ethanol</td>
			<td style="width:185px;">Fisher chemical</td>
			<td style="width:119px;">E/0600DF/15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Filter paper&#160;</td>
			<td style="width:185px;">Whatman</td>
			<td style="width:119px;">10311804</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Surgical tape</td>
			<td style="width:185px;">3M</td>
			<td style="width:119px;">1530-1</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:312px;">CNBr-Activated agarose beads (sepharose 4B)</td>
			<td style="width:185px;">GE Healthcare</td>
			<td style="width:119px;">17-0430-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Sintered glass filter</td>
			<td style="width:185px;">DURAN</td>
			<td style="width:119px;">2515703</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Centrifuge tube 50 mL</td>
			<td style="width:185px;">TPP</td>
			<td style="width:119px;">91050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Purified immunoglobulin G (HsIgG)</td>
			<td style="width:185px;">MP Biomedicals</td>
			<td style="width:119px;">855908</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Rotating shaker</td>
			<td style="width:185px;">IKA</td>
			<td>4016000</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:312px;">NaN3 (sodium azide)</td>
			<td style="width:185px;">Sigma</td>
			<td style="width:119px;">71290</td>
			<td>Toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Quick filtration material (Miracloth)</td>
			<td style="width:185px;">Merk-Millipore</td>
			<td style="width:119px;">475855</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Ultracentrifube XNP-80</td>
			<td style="width:185px;">Beckman Coulter</td>
			<td style="width:119px;">A99839</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Rotor SW 32 Ti</td>
			<td style="width:185px;">Beckman Coulter</td>
			<td style="width:119px;">369650</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Ultracentrifuge tubes</td>
			<td style="width:185px;">Beckman Coulter</td>
			<td style="width:119px;">326823</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Glass teflon homogenizer (Potter)</td>
			<td style="width:185px;">Wheaton</td>
			<td style="width:119px;">357426</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Spin column (Mobicol)</td>
			<td style="width:185px;">Mo Bi Tec</td>
			<td style="width:119px;">M1003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Filter 35&#181;m for spin column (Mobicol)</td>
			<td style="width:185px;">Mo Bi Tec</td>
			<td style="width:119px;">M513515</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">AcTEV</td>
			<td style="width:185px;">Invitrogen</td>
			<td style="width:119px;">12575-015</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Centrifugal evaporator (Speed Vac)</td>
			<td style="width:185px;">Eppendorf</td>
			<td>5305000100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">FBN1A(PGL35) antibody</td>
			<td style="width:185px;">AgriSera</td>
			<td>AS06 116</td>
			<td>RRID:AB_2247012</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sand, Fontainebleau</td>
			<td style="width:185px;">VWR</td>
			<td>27460.295</td>
			<td></td>
		</tr>
	</tbody>
</table>

affinity purification of chloroplast translocon protein complexes using the tap tag

Chloroplast biogenesis requires the import of thousands of nucleus-encoded proteins into the plastid. The import of these proteins depends on the translocon at the outer (TOC) and inner (TIC) chloroplast membranes. The TOC and TIC complexes are multimeric and probably contain yet unknown components. One of the main goals in the field is to establish the complete inventory of TOC and TIC components. For the isolation of TOC-TIC complexes and the identification of new components, the preprotein receptor TOC159 has been modified N-terminally by the addition of the tandem affinity purification (TAP) tag resulting in TAP-TOC159. The TAP-tag is designed for two sequential affinity purification steps (hence "tandem affinity"). The TAP-tag used in these studies consists of a N-terminal IgG-binding domain derived from Staphylococcus aureus Protein A (ProtA) followed by a calmodulin-binding peptide (CBP). Between these two affinity tags, a tobacco etch virus (TEV) protease cleavage site has been included. Therefore, TEV protease can be used for gentle elution of TOC159-containing complexes after binding to IgG beads. In the protocol presented here, the second Calmodulin-affinity purification step was omitted. The purification protocol starts with the preparation and solubilization of total cellular membranes. After the detergent-treatment, the solubilized membrane proteins are incubated with IgG beads for the immunoisolation of TAP-TOC159-containing complexes. Upon binding and extensive washing, TAP-TOC159 containing complexes are cleaved and released from the IgG beads using the TEV protease whereby the S. aureus IgG-binding domain is removed. Western blotting of the isolated TOC159-containing complexes can be used to confirm the presence of known or suspected TOC and TIC proteins. More importantly, the TOC159-containing complexes have been used successfully to identify new components of the TOC and TIC complexes by mass spectrometry. The protocol that we present potentially allows the efficient isolation of any membrane-bound protein complex to be used for the identification of yet unknown components by mass spectrometry.

We here described a protocol for the purification of a TAP-tagged chloroplast envelope protein complex from transgenic A. thaliana plants. As shown in Figure 1A, plants expressing the 35S:NTAP-TOC159 construct were used to isolate this complex while plants expressing the 35S:NTAP construct were used as a control. Figure 1B shows detailed steps of the purification of TAP-TOC159 protein complexes followed by mass spectrometry to allow the identification of interacting proteins. The immunoblot analysis (Figure 2, right side) confirms that isolated TAP-TOC159 interacts with TOC75 and TOC33 of the TOC complex. The chloroplast outer membrane kinase KOC1 known to phosphorylate TOC159 also co-isolated with the TAP-TOC159 complex (Figure 2B, right side). The presence of TIC110 reveals that the isolated TAP-TOC159 complex also contains the components of the TIC complex. The FBN1A antibody raised against a plastoglobule protein unrelated to protein import did not recognize the TAP-TOC159 complex indicating the absence of contaminations. In the negative control, immunoprecipitated NTAP protein did not co-isolate with any of the TOC-TIC proteins (Figure 2, left side) and confirms the specificity of the TAP-TOC159 purification.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58532/58532fig1.jpg" /> 
Figure 1. Scheme of the constructs and affinity purification procedure. (A). The 35S:NTAP-TOC159 construct, encoding NTAP-TOC159 was stably expressed in Arabidopsis thaliana. Negative control plants expressed the 35S:NTAP construct, encoding the TAP-tag alone. (B). The step wise affinity purification protocol consists of membrane isolation and solubilization, IgG agarose immuno-purification, washes, TEV protease cleavage and elution. <a href="https://www.jove.com/files/ftp_upload/58532/58532fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58532/58532fig2.jpg" /> 
Figure 2. Tandem affinity purification of the TOC159 protein. N-terminally TAP-tagged TOC159 was purified from NTAP-TOC15:ppi2 Arabidopsis plants and TAP protein purified from TAP:WT plants was used as a negative control. Total protein extracts (T), solubilized membrane proteins = load fraction (L), flow through (FT), first and last wash fractions (W1 and W6) and 10% of the TEV eluate were analyzed by Western blotting. The membrane was probed with the antibodies against TOC159 (AT4G02510), TOC75 (AT3G46740), TOC33 (AT1G02280), KOC1 (AT4G32250), TIC110 (AT1G06950), and FBN1A (AT4G04020). <a href="https://www.jove.com/files/ftp_upload/58532/58532fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Table of buffers</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Coupling Buffer</td>
			<td>NaHCO3 
			Adjust to pH 8.5 adjust pH with 0.1 M Na2CO3</td>
			<td>100 mM 
			&#160;</td>
		</tr>
		<tr>
			<td>Blocking Buffer</td>
			<td>Tris&#8211;HCl 
			Adjust to pH 8 with HCl.</td>
			<td>100 mM 
			&#160;</td>
		</tr>
		<tr>
			<td>PBS Buffer</td>
			<td>Na2HPO4 
			KH2PO4 
			NaCl 
			KCl 
			Adjust to pH 7.3 with HCl.</td>
			<td>4.3 mM 
			1.4 mM 
			137 
			2.7 
			&#160;</td>
		</tr>
		<tr>
			<td>NaCl coupling Buffer</td>
			<td>Coupling buffer 
			NaCl</td>
			<td>&#160; 
			1 M</td>
		</tr>
		<tr>
			<td>2X grinding Buffer</td>
			<td>Tris-HCl, pH 7.5 with HCl. 
			NaCl 
			NaF 
			PMSF 
			Plant protease inhibitor cocktail</td>
			<td>100 mM 
			200 mM 
			5 mM 
			1 mM 
			0.20%</td>
		</tr>
		<tr>
			<td>4X solubilisation solution</td>
			<td>Glycerol 
			Triton-X100</td>
			<td>40% 
			3%</td>
		</tr>
		<tr>
			<td>Buffer A</td>
			<td>&#160; 
			2X grinding Buffer 
			1X grinding Buffer 
			4X solubilisation solution</td>
			<td>100 ml 
			50 ml 
			25 ml 
			25%</td>
		</tr>
		<tr>
			<td>TEV elution Buffer</td>
			<td>Tris-HCl, pH 8 with HCl 
			EDTA, pH 8 with HCl 
			NaCl 
			Glycerol 
			Triton-X100 
			DTT</td>
			<td>50 mM 
			0.5 mM 
			100 mM 
			10% 
			0.75% 
			1 mM</td>
		</tr>
		<tr>
			<td>2x SDS-PAGE loading Buffer</td>
			<td>Tris-HCl pH 6.8 with HCl 
			SDS 
			Glycerol 
			Bromophenol blue 
			DTT</td>
			<td>0.1 M 
			4% 
			20% 
			0.20% 
			0.2 M</td>
		</tr>
	</tbody>
</table>
Table 1. Buffer recipes.

Watch this Scientific Journal Video about Affinity Purification of Chloroplast Translocon Protein Complexes Using the TAP Tag at JoVE.com

Affinity Purification of Chloroplast Translocon Protein Complexes Using the TAP Tag

We here present a proven and tested protocol for the purification of chloroplast protein import complexes (TOC-TIC complex) using the TAP-tag. The one-step affinity-isolation protocol can potentially be applied to any protein and be used to identify new interaction partners by mass spectrometry.

Chloroplast biogenesis requires the import of thousands of nucleus-encoded proteins into the plastid. The import of these proteins depends on the ...

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10.1K Views.  University of Neuchatel. We here present a proven and tested protocol for the purification of chloroplast protein import complexes (TOC-TIC complex) using the TAP-tag. The one-step affinity-isolation protocol can potentially be applied to any protein and be used to identify new interaction partners by mass spectrometry.

Video: Affinity Purification of Chloroplast Translocon Protein Complexes Using the TAP Tag

Isolation of Cognate RNA-protein Complexes from Cells Using Oligonucleotide-directed Elution

Ribonucleoprotein particles direct the biogenesis and post-transcriptional regulation of all mRNAs through distinct combinations of RNA binding proteins. They are composed of position-dependent, cis-acting RNA elements and unique combinations of RNA binding proteins. Defining the composition of a specific RNP is essential to achieving a fundamental understanding of gene regulation. The isolation of a select RNP is akin to finding a needle in a haystack. Here, we demonstrate an approach to isolate RNPs associated at the 5' untranslated region of a select mRNA in asynchronous, transfected cells. This cognate RNP has been demonstrated to be necessary for the translation of select viruses and cellular stress-response genes.
The demonstrated RNA-protein co-precipitation protocol is suitable for the downstream analysis of protein components through proteomic analyses, immunoblots, or suitable biochemical identification assays. This experimental protocol demonstrates that DHX9/RNA helicase A is enriched at the 5' terminus of cognate retroviral RNA and provides preliminary information for the identification of its association with cell stress-associated huR and junD cognate mRNAs.

This manuscript describes an approach to isolate select cognate RNPs formed in eukaryotic cells via a specific oligonucleotide-directed enrichment. We demonstrate the applicability of this approach by isolating a cognate RNP bound to the retroviral 5' untranslated region that is composed of DHX9/RNA helicase A.

Ribonucleoprotein particles direct the biogenesis and post-transcriptional regulation of all mRNAs through distinct combinations of RNA binding proteins. ...

Protein Complex Affinity Capture from Cryomilled Mammalian Cells

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this technique is also frequently referred to as immunoprecipitation. Affinity capture can be applied in a bench-scale and in a high-throughput context. When coupled with protein mass spectrometry, affinity capture has proven to be a workhorse of interactome analysis. Although there are potentially many ways to execute the numerous steps involved, the following protocols implement our favored methods. Two features are distinctive: the use of cryomilled cell powder to produce cell extracts, and antibody-coupled paramagnetic beads as the affinity medium. In many cases, we have obtained superior results to those obtained with more conventional affinity capture practices. Cryomilling avoids numerous problems associated with other forms of cell breakage. It provides efficient breakage of the material, while avoiding denaturation issues associated with heating or foaming. It retains the native protein concentration up to the point of extraction, mitigating macromolecular dissociation. It reduces the time extracted proteins spend in solution, limiting deleterious enzymatic activities, and it may reduce the non-specific adsorption of proteins by the affinity medium. Micron-scale magnetic affinity media have become more commonplace over the last several years, increasingly replacing the traditional agarose- and Sepharose-based media. Primary benefits of magnetic media include typically lower non-specific protein adsorption; no size exclusion limit because protein complex binding occurs on the bead surface rather than within pores; and ease of manipulation and handling using magnets.

Here we describe protocols to disrupt mammalian cells by solid-state milling at a cryogenic temperature, produce a cell extract from the resulting cell powder, and isolate protein complexes of interest by affinity capture upon antibody-coupled micron-scale paramagnetic beads.

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this ...

Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo identification of novel subunits and post-translational modifications. Bacterial systems allow for the expression and purification of a wide variety of single polypeptides and protein complexes. However, this system does not enable the purification of protein subunits that contain post-translational modifications (e.g., phosphorylation and acetylation), and the identification of novel regulatory subunits that are only present/expressed in the eukaryotic system. Here, we provide a detailed description of a novel, robust, and efficient tandem affinity purification (TAP) method using STREP- and FLAG-tagged proteins that facilitates the purification of protein complexes with transiently or stably expressed epitope-tagged proteins from eukaryotic cells. This protocol can be applied to characterize protein complex functionality, to discover post-translational modifications on complex subunits, and to identify novel regulatory complex components by mass spectrometry. Notably, this TAP method can be applied to study protein complexes formed by eukaryotic or pathogenic (viral and bacterial) components, thus yielding a wide array of downstream experimental opportunities. We propose that researchers working with protein complexes could utilize this approach in many different ways.

We describe here a novel, robust, and efficient tandem affinity purification (TAP) method for the expression, isolation, and characterization of protein complexes from eukaryotic cells. This protocol could be utilized for the biochemical characterization of discrete complexes as well as the identification of novel interactors and post-translational modifications that regulate their function.

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo ...

Identification of Plant Ice-binding Proteins Through Assessment of Ice-recrystallization Inhibition and Isolation Using Ice-affinity Purification

Ice-binding proteins (IBPs) belong to a family of stress-induced proteins that are synthesized by certain organisms exposed to subzero temperatures. In plants, freeze damage occurs when extracellular ice crystals grow, resulting in the rupture of plasma membranes and possible cell death. Adsorption of IBPs to ice crystals restricts further growth by a process known as ice-recrystallization inhibition (IRI), thereby reducing cellular damage. IBPs also demonstrate the ability to depress the freezing point of a solution below the equilibrium melting point, a property known as thermal hysteresis (TH) activity. These protective properties have raised interest in the identification of novel IBPs due to their potential use in industrial, medical and agricultural applications. This paper describes the identification of plant IBPs through 1) the induction and extraction of IBPs in plant tissue, 2) the screening of extracts for IRI activity, and 3) the isolation and purification of IBPs. Following the induction of IBPs by low temperature exposure, extracts are tested for IRI activity using a &#39;splat assay&#39;, which allows the observation of ice crystal growth using a standard light microscope. This assay requires a low protein concentration and generates results that are quickly obtained and easily interpreted, providing an initial screen for ice binding activity. IBPs can then be isolated from contaminating proteins by utilizing the property of IBPs to adsorb to ice, through a technique called &#39;ice-affinity purification&#39;. Using cell lysates collected from plant extracts, an ice hemisphere can be slowly grown on a brass probe. This incorporates IBPs into the crystalline structure of the polycrystalline ice. Requiring no a priori biochemical or structural knowledge of the IBP, this method allows for recovery of active protein. Ice-purified protein fractions can be used for downstream applications including the identification of peptide sequences by mass spectrometry and the biochemical analysis of native proteins.

This paper outlines the identification of ice-binding proteins from freeze-tolerant plants through the assessment of ice-recrystallization inhibition activity and subsequent isolation of native IBPs using ice-affinity purification.

Ice-binding proteins (IBPs) belong to a family of stress-induced proteins that are synthesized by certain organisms exposed to subzero temperatures. In ...

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

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

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

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

Detection and Visualization of DNA Damage-induced Protein Complexes in Suspension Cell Cultures Using the Proximity Ligation Assay

The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of programmed DNA breaks in lymphocytes. The Ataxia-Telangiectasia Mutated kinase (ATM), ATM- and Rad3-Related kinase (ATR) and the catalytic subunit of DNA-dependent Protein Kinase (DNA-PKcs) are among the first that are activated upon induction of DNA damage, and are central regulators of a network that controls DNA repair, apoptosis and cell survival. As part of a tumor-suppressive pathway, ATM and ATR activate p53 through phosphorylation, thereby regulating the transcriptional activity of p53. DNA damage also results in the formation of so-called ionizing radiation-induced foci (IRIF) that represent complexes of DNA damage sensor and repair proteins that accumulate at the sites of DNA damage, which are visualized by fluorescence microscopy. Co-localization of proteins in IRIFs, however, does not necessarily imply direct protein-protein interactions, as the resolution of fluorescence microscopy is limited. 
In situ Proximity Ligation Assay (PLA) is a novel technique that allows the direct visualization of protein-protein interactions in cells and tissues with unprecedented specificity and sensitivity. This technique is based on the spatial proximity of specific antibodies binding to the proteins of interest. When the interrogated proteins are within ~40 nm an amplification reaction is triggered by oligonucleotides that are conjugated to the antibodies, and the amplification product is visualized by fluorescent labeling, yielding a signal that corresponds to the subcellular location of the interacting proteins. Using the established functional interaction between ATM and p53 as an example, it is demonstrated here how PLA can be used in suspension cell cultures to study the direct interactions between proteins that are integral parts of the DNA damage response.

Here, it is demonstrated how the in situ Proximity Ligation Assay (PLA) can be used to detect and visualize the direct protein-protein interactions between ATM and p53 in suspension cell cultures exposed to genotoxic stress.

The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of ...

Rab10 Phosphorylation Detection by LRRK2 Activity Using SDS-PAGE with a Phosphate-binding Tag

Mutations in leucine-rich repeat kinase 2 (LRRK2) have been shown to be linked with familial Parkinson&#39;s disease (FPD). Since abnormal activation of the kinase activity of LRRK2 has been implicated in the pathogenesis of PD, it is essential to establish a method to evaluate the physiological levels of the kinase activity of LRRK2. Recent studies revealed that LRRK2 phosphorylates members of the Rab GTPase family, including Rab10, under physiological conditions. Although the phosphorylation of endogenous Rab10 by LRRK2 in cultured cells could be detected by mass spectrometry, it has been difficult to detect it by immunoblotting due to the poor sensitivity of currently available phosphorylation-specific antibodies for Rab10. Here, we describe a simple method of detecting the endogenous levels of Rab10 phosphorylation by LRRK2 based on immunoblotting utilizing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) combined with a phosphate-binding tag (P-tag), which is N-(5-(2-aminoethylcarbamoyl)pyridin-2-ylmetyl)-N,N&#39;,N&#39;-tris(pyridin-2-yl-methyl)-1,3-diaminopropan-2-ol. The present protocol not only provides an example of the methodology utilizing the P-tag but also enables the assessment of how mutations as well as inhibitor treatment/administration or any other factors alter the downstream signaling of LRRK2 in cells and tissues.

The present study describes a simple method of detecting endogenous levels of Rab10 phosphorylation by leucine-rich repeat kinase 2.

Mutations in leucine-rich repeat kinase 2 (LRRK2) have been shown to be linked with familial Parkinson&#39;s disease (FPD). Since abnormal activation of ...

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

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of protein-protein interactions (PPI) is no novelty, experimental approaches aiming to characterize endogenous protein-metabolite interactions (PMI) constitute a rather recent development. Herein, we present a protocol that allows simultaneous characterization of the PPI and PMI of a protein of choice, referred to as bait. Our protocol was optimized for Arabidopsis cell cultures and combines affinity purification (AP) with mass spectrometry (MS)-based protein and metabolite detection. In short, transgenic Arabidopsis lines, expressing bait protein fused to an affinity tag, are first lysed to obtain a native cellular extract. Anti-tag antibodies are used to pull down protein and metabolite partners of the bait protein. The affinity-purified complexes are extracted using a one-step methyl tert-butyl ether (MTBE)/methanol/water method. Whilst metabolites separate into either the polar or the hydrophobic phase, proteins can be found in the pellet. Both metabolites and proteins are then analyzed by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS or UPLC-MS/MS). Empty-vector (EV) control lines are used to exclude false positives. The major advantage of our protocol is that it enables identification of protein and metabolite partners of a target protein in parallel in near-physiological conditions (cellular lysate). The presented method is straightforward, fast, and can be easily adapted to biological systems other than plant cell cultures.

Protein-protein and protein-metabolite interactions are crucial for all cellular functions. Herein, we describe a protocol that allows parallel analysis of these interactions with a protein of choice. Our protocol was optimized for plant cell cultures and combines affinity purification with mass spectrometry-based protein and metabolite detection.

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

Preparation of Chloroplast Sub-compartments from Arabidopsis for the Analysis of Protein Localization by Immunoblotting or Proteomics

Chloroplasts are major components of plant cells. Such plastids fulfill many crucial functions, such as assimilation of carbon, sulfur and nitrogen as well as synthesis of essential metabolites. These organelles consist of the following three key sub-compartments. The envelope, characterized by two membranes, surrounds the organelle and controls the communication of the plastid with other cell compartments. The stroma is the soluble phase of the chloroplast and the main site where carbon dioxide is converted into carbohydrates. The thylakoid membrane is the internal membrane network consisting of grana (flat compressed sacs) and lamellae (less dense structures), where oxygenic photosynthesis takes place. The present protocol describes step by step procedures required for the purification, using differential centrifugations and Percoll gradients, of intact chloroplasts from Arabidopsis, and their fractionation, using sucrose gradients, in three sub-compartments (i.e., envelope, stroma, and thylakoids). This protocol also provides instructions on how to assess the purity of these fractions using markers associated to the various chloroplast sub-compartments. The method described here is valuable for subplastidial localization of proteins using immunoblotting, but also for subcellular and subplastidial proteomics and other studies.

Here, we describe a method to purify intact chloroplasts from Arabidopsis leaves and their three main sub-compartments (envelope, stroma, and thylakoids), using a combination of differential centrifugations, continuous Percoll gradients, and discontinuous sucrose gradients. The method is valuable for subplastidial and subcellular localization of proteins by immunoblotting and proteomics.

Chloroplasts are major components of plant cells. Such plastids fulfill many crucial functions, such as assimilation of carbon, sulfur and nitrogen as ...

Single-step Purification of Macromolecular Complexes Using RNA Attached to Biotin and a Photo-cleavable Linker

For many years, the exceptionally strong and rapidly formed interaction between biotin and streptavidin has been successfully utilized for partial purification of biologically important RNA/protein complexes. However, this strategy suffers from one major disadvantage that limits its broader utilization: the biotin/streptavidin interaction can be broken only under denaturing conditions that also disrupt the integrity of the eluted complexes, hence precluding their subsequent functional analysis and/or further purification by other methods. In addition, the eluted samples are frequently contaminated with the background proteins that nonspecifically associate with streptavidin beads, complicating the analysis of the purified complexes by silver staining and mass spectrometry. To overcome these limitations, we developed a variant of the biotin/streptavidin strategy in which biotin is attached to an RNA substrate via a photo-cleavable linker and the complexes immobilized on streptavidin beads are selectively eluted to solution in a native form by long wave UV, leaving the background proteins on the beads. Shorter RNA binding substrates can be synthesized chemically with biotin and the photo-cleavable linker covalently attached to the 5&#39; end of the RNA, whereas longer RNA substrates can be provided with the two groups by a complementary oligonucleotide. These two variants of the UV-elution method were tested for purification of the U7 snRNP-dependent processing complexes that cleave histone pre-mRNAs at the 3&#39; end and they both proved to compare favorably to other previously developed purification methods. The UV-eluted samples contained readily detectable amounts of the U7 snRNP that was free of major protein contaminants and suitable for direct analysis by mass spectrometry and functional assays. The described method can be readily adapted for purification of other RNA binding complexes and used in conjunction with single- and double-stranded DNA binding sites to purify DNA-specific proteins and macromolecular complexes.

RNA/protein complexes purified using botin-streptavidin strategy are eluted to solution under denaturing conditions in a form unsuitable for further purification and functional analysis. Here, we describe a modification of this strategy that utilizes a photo-cleavable linker in RNA and a gentle UV-elution step, yielding native and fully functional RNA/protein complexes.

Affinity Purification of Chloroplast Translocon Protein Complexes Using the TAP Tag

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