Name: isolation of labile multi-protein complexes by in vivo controlled cellular cross-linking and immuno-magnetic affinity chromatography
Uploaded: 2010-03-09T17:05:05
Description: Watch this Scientific Journal Video about Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography at JoVE.com

This work was supported by grants from the National Institutes of Health to V.F. (NS42599 and GM077569).

1. Preparing for Crosslinking
<ol>
	<li>You will need to plate a sufficient number of cells to allow isolation of 500 &mu;g of protein per standard tube reaction. A single tube may be enough for identification of putative interactors of a protein of interest by immunoblot. In this case bead bound material can be eluted with SDS-PAGE sample buffer (immunoprecipitation). For mass spectrometry analysis, the number of standard reactions should be increased at least ten times and protein complexes should be eluted by out-...

<ol>
	<li>Salazar, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hermansky-Pudlak+syndrome+protein+complexes+associate+with+phosphatidylinositol+4-kinase+type+II+alpha+in+neuronal+and+non-neuronal+cells.">Hermansky-Pudlak syndrome protein complexes associate with phosphatidylinositol 4-kinase type II alpha in neuronal and non-neuronal cells.</a> J Biol Chem. 284, 1790-1802 (2009).</li><li>Lomant, A. J., Fairbanks, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+probes+of+extended+biological+structures:+synthesis+and+properties+of+the+cleavable+protein+cross-linking+reagent+[35S]dithiobis(succinimidyl+propionate.">Chemical probes of extended biological structures: synthesis and properties of the cleavable protein cross-linking reagent [35S]dithiobis(succinimidyl propionate.</a> J Mol Biol. 104, 243-261 (1976).</li><li>Xiang, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+DSP,+a+reversible+cross-linker,+to+fix+tissue+sections+for+immunostaining,+microdissection+and+expression+profiling.">Using DSP, a reversible cross-linker, to fix tissue sections for immunostaining, microdissection and expression profiling.</a> Nucleic Acids Res. 32, e185-e185 (2004).</li><li>Craige, B., Salazar, G., Faundez, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphatidylinositol-4-Kinase+Type+II+Alpha+Contains+an+AP-3+Sorting+Motif+and+a+Kinase+Domain+that+are+both+Required+for+Endosome+Traffic.">Phosphatidylinositol-4-Kinase Type II Alpha Contains an AP-3 Sorting Motif and a Kinase Domain that are both Required for Endosome Traffic.</a> Mol Biol Cell. 19, 1415-1426 (2008).</li><li>Newell-Litwa, K., Seong, E., Burmeister, M., Faundez, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+and+non-neuronal+functions+of+the+AP-3+sorting+machinery.">Neuronal and non-neuronal functions of the AP-3 sorting machinery.</a> J Cell Sci. 120, 531-541 (2007).</li><li>Salazar, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Zinc+Transporter+ZnT3+interacts+with+AP-3+and+it+is+Targeted+to+a+Distinct+Synaptic+Vesicle+Subpopulation.">The Zinc Transporter ZnT3 interacts with AP-3 and it is Targeted to a Distinct Synaptic Vesicle Subpopulation.</a> Mol Biol Cell. 15, 575-587 (2004).</li><li>Trinkle-Mulcahy, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identifying+specific+protein+interaction+partners+using+quantitative+mass+spectrometry+and+bead+proteomes.">Identifying specific protein interaction partners using quantitative mass spectrometry and bead proteomes.</a> J Cell Biol. 183, 223-239 (2008).</li></ol>

DSP, a membrane-permeable, chemically reducible crosslinker with a spacer arm of 12 &Aring; is used to stabilize transient protein interactions 1,2,3,4. Here we exemplified this strategy with the adaptor complex AP-3 a soluble protein complex that recognizes and sorts membrane proteins into vesicles from endosomes 5. AP-3 selectively binds to the zinc transporter ZnT3 and the lipid kinase phosphatidylinositol-4-kinase type II alpha but not transferrin receptor 1,4,6. We expanded these obs...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Phosphate Buffered Saline</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>P4417</td>
<td>Dissolve 1 tablet in 200 mL water; add MgCl2 to a final concentration of 1 mM and CaCl2 to a final concentration of 0.1 mM</td>
</tr>
<tr>
<td>Dithiobis (succinimidyl propionate) (DSP)</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>22585</td>
<td>Moisture sensitive, store in air tight container 4&deg;C</td>
</tr>
<tr>
<td>Dimethyl sulphoxide (DMSO) Hybri-Max</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>D2650</td>
<td>&nbsp;</td>
<tr>
<td>Triton X-100, SigmaUltra</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>T9284</td>
<td>&nbsp;</td>
<tr>
<td>Dynabeads, Sheep anti-Mouse IgG</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>110.31</td>
<td>Beads are also available as sheep anti-rabbit</td>
</tr>
<tr>
<td>Dyna-Mag-2 magnet</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>123-21D</td>
<td>&nbsp;</td>		</tbody>
		</table>
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isolation of labile multi-protein complexes by in vivo controlled cellular cross-linking and immuno-magnetic affinity chromatography

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude stringent methods for the isolation and identification of protein networks around a protein of interest. The use of chemical crosslinkers allows the selective stabilization of labile interactions, thus bypassing biochemical limitations for purification. Here we present a protocol amenable for cells in culture that uses a homobifunctional crosslinker with a spacer arm of 12 &Aring;, dithiobis-(succinimidyl proprionate) (DSP). DSP is cleaved by reduction of a disulphide bond present in the molecule. Cross-linking combined with immunoaffinity chromatography of proteins of interest with magnetic beads allows the isolation of protein complexes that otherwise would not withstand purification. This protocol is compatible with regular western blot techniques and it can be scaled up for protein identification by mass spectrometry1.
Stephanie A. Zlatic and Pearl V. Ryder contributed equally to this work.

Watch this Scientific Journal Video about Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography at JoVE.com

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The cell permeable crosslinker DSP [dithiobis-(succinimidyl propionate)] stabilizes transient and labile interactions in vivo, which allows their isolation using stringent protein complex purification techniques. Here we present a technique for crosslinking cells grown in culture followed by isolation of protein complexes by immunoprecipitation.

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude ...

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