Name: affinity purification of chloroplast translocon protein complexes using the tap tag
Uploaded: 2018-11-01T15:00:00.000+00:00
Duration: 7 min 1 s
Description: Watch this Scientific Journal Video about Affinity Purification of Chloroplast Translocon Protein Complexes Using the TAP Tag at JoVE.com

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. 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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><tbody><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>PanReac&amp;nbsp; AppliChem</td><td>A0384</td><td></td></tr><tr><td>Tris</td><td>Biosolve</td><td>0020092391BS</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>S7909</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>PanReac&amp;nbsp; AppliChem</td><td>A2946</td><td></td></tr><tr><td>NaCl</td><td>PanReac&amp;nbsp; AppliChem</td><td>A2942</td><td></td></tr><tr><td>NaF</td><td>Sigma</td><td>S1509</td><td>Toxic</td></tr><tr><td>PMSF</td><td>PanReac&amp;nbsp; AppliChem</td><td>A0999</td><td>Toxic</td></tr><tr><td>Plant protease inhibitor cocktail</td><td>Sigma</td><td>P9599</td><td></td></tr><tr><td>Glycerol</td><td>PanReac&amp;nbsp; AppliChem</td><td>A0970</td><td></td></tr><tr><td>Triton X100</td><td>Roche</td><td>10789704001</td><td></td></tr><tr><td>Glycine</td><td>PanReac&amp;nbsp; AppliChem</td><td>A1067</td><td></td></tr><tr><td>EDTA</td><td>Carl Roth</td><td>8043</td><td></td></tr><tr><td>Dithiothreitol (DTT)</td><td>PanReac&amp;nbsp; AppliChem</td><td>A1101</td><td></td></tr><tr><td>SDS</td><td>PanReac&amp;nbsp; AppliChem</td><td>A0675</td><td></td></tr><tr><td>Bromophenol blue</td><td>Sigma</td><td>B0126</td><td></td></tr><tr><td>HCl</td><td>VWR</td><td>20252-290</td><td>Corrosive</td></tr><tr><td>Sucrose</td><td>PanReac&amp;nbsp; AppliChem</td><td>A3935</td><td></td></tr><tr><td>Murashige and Skoog medium with vitamins</td><td>Duchefa Biochemie</td><td>M0222</td><td></td></tr><tr><td>Phytoagar</td><td>Duchefa Biochemie</td><td>P1003</td><td></td></tr><tr><td>Petri plate</td><td>Greiner Bio-one</td><td>639102</td><td></td></tr><tr><td>1.5 mL microfuge tubes</td><td>Sarstedt</td><td>72.706</td><td></td></tr><tr><td>Ethanol</td><td>Fisher chemical</td><td>E/0600DF/15</td><td></td></tr><tr><td>Filter paper&amp;nbsp;</td><td>Whatman</td><td>10311804</td><td></td></tr><tr><td>Surgical tape</td><td>3M</td><td>1530-1</td><td></td></tr><tr><td>CNBr-Activated agarose beads (sepharose 4B)</td><td>GE Healthcare</td><td>17-0430-01</td><td></td></tr><tr><td>Sintered glass filter</td><td>DURAN</td><td>2515703</td><td></td></tr><tr><td>Centrifuge tube 50 mL</td><td>TPP</td><td>91050</td><td></td></tr><tr><td>Purified immunoglobulin G (HsIgG)</td><td>MP Biomedicals</td><td>855908</td><td></td></tr><tr><td>Rotating shaker</td><td>IKA</td><td>4016000</td><td></td></tr><tr><td>NaN&lt;sub&gt;3 &lt;/sub&gt;(sodium azide)</td><td>Sigma</td><td>71290</td><td>Toxic</td></tr><tr><td>Quick filtration material (Miracloth)</td><td>Merk-Millipore</td><td>475855</td><td></td></tr><tr><td>Ultracentrifube XNP-80</td><td>Beckman Coulter</td><td>A99839</td><td></td></tr><tr><td>Rotor SW 32 Ti</td><td>Beckman Coulter</td><td>369650</td><td></td></tr><tr><td>Ultracentrifuge tubes</td><td>Beckman Coulter</td><td>326823</td><td></td></tr><tr><td>Glass teflon homogenizer (Potter)</td><td>Wheaton</td><td>357426</td><td></td></tr><tr><td>Spin column (Mobicol)</td><td>Mo Bi Tec</td><td>M1003</td><td></td></tr><tr><td>Filter 35&amp;micro;m for spin column (Mobicol)</td><td>Mo Bi Tec</td><td>M513515</td><td></td></tr><tr><td>AcTEV</td><td>Invitrogen</td><td>12575-015</td><td></td></tr><tr><td>Centrifugal evaporator (Speed Vac)</td><td>Eppendorf</td><td>5305000100</td><td></td></tr><tr><td>FBN1A(PGL35) antibody</td><td>AgriSera</td><td>AS06 116</td><td>RRID:AB_2247012</td></tr><tr><td>Sand, Fontainebleau</td><td>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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10.2K 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

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 ...

Method for Efficient Refolding and Purification of Chemoreceptor Ligand Binding Domain

Identification of natural ligands of chemoreceptors and structural studies aimed at elucidation of the molecular basis of the ligand specificity can be greatly facilitated by the production of milligram amounts of pure, folded ligand binding domains. Attempts to heterologously express periplasmic ligand binding domains of bacterial chemoreceptors in Escherichia coli (E. coli) often result in their targeting into inclusion bodies. Here, a method is presented for protein recovery from inclusion bodies, its refolding and purification, using the periplasmic dCACHE ligand binding domain of Campylobacter jejuni (C. jejuni) chemoreceptor Tlp3 as an example. The approach involves expression of the protein of interest with a cleavable His6-tag, isolation and urea-mediated solubilisation of inclusion bodies, protein refolding by urea depletion, and purification by means of affinity chromatography, followed by tag removal and size-exclusion chromatography. The circular dichroism spectroscopy is used to confirm the folded state of the pure protein. It has been demonstrated that this protocol is generally useful for production of milligram amounts of dCACHE periplasmic ligand binding domains of other bacterial chemoreceptors in a soluble and crystallisable form.

A procedure is presented for the refolding of the dCACHE periplasmic ligand binding domain of Campylobacter jejuni chemoreceptor Tlp3 from inclusion bodies and the purification to yield milligram quantities of protein.

Identification of natural ligands of chemoreceptors and structural studies aimed at elucidation of the molecular basis of the ligand specificity can be ...

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 ...

Isolation of Physiologically Active Thylakoids and Their Use in Energy-Dependent Protein Transport Assays

Chloroplasts are the organelles in green plants responsible for carrying out numerous essential metabolic pathways, most notably photosynthesis. Within the chloroplasts, the thylakoid membrane system houses all the photosynthetic pigments, reaction center complexes, and most of the electron carriers, and is responsible for light-dependent ATP synthesis. Over 90% of chloroplast proteins are encoded in the nucleus, translated in the cytosol, and subsequently imported into the chloroplast. Further protein transport into or across the thylakoid membrane utilizes one of four translocation pathways. Here, we describe a high-yield method for isolation of transport-competent thylakoids from peas (Pisum sativum), along with transport assays through the three energy-dependent cpTat, cpSec1, and cpSRP-mediated pathways. These methods enable experiments relating to thylakoid protein localization, transport energetics, and the mechanisms of protein translocation across biological membranes.

We present protocols herein for high-yield isolation of physiologically active thylakoids and protein transport assays for the chloroplast twin arginine translocation (cpTat), secretory (cpSec1), and signal recognition particle (cpSRP) pathways.

Chloroplasts are the organelles in green plants responsible for carrying out numerous essential metabolic pathways, most notably photosynthesis. Within ...

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 ...

Biology

Optimizing FPLC for Efficient Purification of His-Tagged FEN1 Protein

Affinity Purification of a 6X-His-Tagged Protein using a Fast Protein Liquid Chromatography System

Functional characterization of proteins requires them to be expressed and purified in substantial amounts with high purity to perform biochemical assays. The Fast Protein Liquid Chromatography (FPLC) system allows high-resolution separation of complex protein mixtures. By adjusting various parameters in FPLC, such as selecting the appropriate purification matrix, regulating the protein sample's temperature, and managing the sample's flow rate onto the matrix and the elution rate, it is possible to ensure the protein's stability and functionality. In this protocol, we will demonstrate the versatility of the FPLC system to purify 6X-His-tagged flap endonuclease 1 (FEN1) protein, produced in bacterial cultures. To improve protein purification efficiency, we will focus on multiple considerations, including proper column packing and preparation, sample injection using a sample loop, flow rate of sample application to the column, and sample elution parameters. Finally, the chromatogram will be analyzed to identify fractions containing high yields of protein and considerations for proper recombinant protein long-term storage. Optimizing protein purification methods is crucial for improving the precision and reliability of protein analysis.

Author Spotlight: Optimizing Affinity Chromatography for His-Tagged FEN1 Protein

This article provides a procedure for the affinity purification of a human recombinant protein, flap endonuclease 1 (FEN1), which has been labeled with a 6X-histidine tag. The protocol involves the utilization of two distinct immobilized metal ion columns for the purification of the tagged protein.

Functional characterization of proteins requires them to be expressed and purified in substantial amounts with high purity to perform biochemical assays. ...

Identification of Protein Interacting Partners Using Tandem Affinity Purification

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein partners. There are many approaches that allow the identification of interacting partners, including the yeast two hybrid system, as well as pull down assays using recombinant proteins and immunoprecipitation of endogenous proteins followed by mass spectrometry identification1. Recent studies have highlighted the utility of double-affinity tag mediated purification, coupled with two specific elution steps in the identification of interacting proteins. This approach, termed Tandem Affinity Purification (TAP), was initially used in yeast2,3 but more recently has been adapted to use in mammalian cells4-8.
As proof-of-concept we have established a tandem affinity purification (TAP) method using the well-characterized eukaryotic translation initiation factor eIF4E9,10.The cellular translation factor eIF4E is a critical component of the cellular eIF4F complex involved in cap-dependent translation initiation10. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence8. To forgo the need for the generation of clonal cell lines, we developed a rapid system that relies on the expression of the TAP-tagged bait protein from an episomally maintained plasmid based on pMEP4 (Invitrogen). Expression of tagged murine eIF4E from this plasmid was controlled using the cadmium chloride inducible metallothionein promoter.
Lysis of the expressing cells and subsequent affinity purification via binding to rabbit IgG agarose, TEV protease cleavage, binding to streptavidin linked agarose and subsequent biotin elution identified numerous proteins apparently specific to the eIF4E pull-down (when compared to control cell lines expressing the TAP tag alone). The identities of the proteins were obtained by excision of the bands from 1D SDS-PAGE and subsequent tandem mass spectrometry. The identified components included the known eIF4E binding proteins eIF4G and 4EBP-1. In addition, other components of the eIF4F complex, of which eIF4E is a component were identified, namely eIF4A and Poly-A binding protein. The ability to identify not only known direct binding partners as well as secondary interacting proteins, further highlights the utility of this approach in the characterization of proteins of unknown function.

Tandem affinity purification is a robust approach for the identification of protein binding partners. As proof of concept, this methodology was applied to the well-characterized translation initiation factor eIF4E to co-precipitate the host cell factors involved in translation initiation. This method is easily adapted to any cellular or viral protein.

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein ...

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification (TAP)

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. However, the use of biochemical screens aimed at extending the knowledge gained from genetic analysis was explored only recently. Here we describe a method to purify the protein complex that associates with any protein of interest from adult fly heads. This method takes advantage of the Drosophila GAL4/UAS system to express a bait protein fused with a Tandem Affinity Purification (TAP) tag in fly neurons in vivo, and then implements two rounds of purification using a TAP procedure similar to the one originally established in yeast1 to purify the interacting protein complex. At the end of this procedure, a mixture of multiple protein complexes is obtained whose molecular identities can be determined by mass spectrometry. Validation of the candidate proteins will benefit from the resource and ease of performing loss-of-function studies in flies. Similar approaches can be applied to other fly tissues. We believe that the combination of genetic manipulations and this proteomic approach in the fly model system holds tremendous potential for tackling fundamental problems in the field of neurobiology and beyond.

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification TAP

Drosophila is famous for its powerful genetic manipulation, but not for its suitability of in-depth biochemical analysis. Here we present a TAP-based procedure to identify interacting partners of any protein of interest from the fly brain. This procedure can potentially lead to new avenues of research.

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. ...

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

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

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

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

Inducible LAP-tagged Stable Cell Lines for Investigating Protein Function, Spatiotemporal Localization and Protein Interaction Networks

Multi-protein complexes, rather than single proteins acting in isolation, often govern molecular pathways regulating cellular homeostasis. Based on this principle, the purification of critical proteins required for the functioning of these pathways along with their native interacting partners has not only allowed the mapping of the protein constituents of these pathways, but has also provided a deeper understanding of how these proteins coordinate to regulate these pathways. Within this context, understanding a protein's spatiotemporal localization and its protein-protein interaction network can aid in defining its role within a pathway, as well as how its misregulation may lead to disease pathogenesis. To address this need, several approaches for protein purification such as tandem affinity purification (TAP) and localization and affinity purification (LAP) have been designed and used successfully. Nevertheless, in order to apply these approaches to pathway-scale proteomic analyses, these strategies must be supplemented with modern technological developments in cloning and mammalian stable cell line generation. Here, we describe a method for generating LAP-tagged human inducible stable cell lines for investigating protein subcellular localization and protein-protein interaction networks. This approach has been successfully applied to the dissection of multiple cellular pathways including cell division and is compatible with high-throughput proteomic analyses.

We describe a method for generating localization and affinity purification (LAP)-tagged inducible stable cell lines for investigating protein function, spatiotemporal subcellular localization and protein-protein interaction networks.

Multi-protein complexes, rather than single proteins acting in isolation, often govern molecular pathways regulating cellular homeostasis. Based on this ...