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

Bu çalışma, VF için Ulusal Sağlık Enstitüleri (NS42599 ve GM077569) hibe ile desteklendi.

1. Crosslinking için hazırlık <ol><li> 500 mg standart tüp reaksiyonu başına protein izolasyonu izin plaka için yeterli sayıda hücre gerekecektir. Bir tek tüp, immunoblotting faiz bir protein varsayılan interactors tanımlama için yeterli olabilir. Bu durumda, boncuk bağlı malzeme, SDS-PAGE örnek tamponu (immunoprecipitation) ile yıkandı olabilir. Kütle spektrometresi analizi için, standart reaksiyonlar sayısı en az on kat artmış olmalı ve protein kompleksleri kullanılan antikor (immünoafinit...

<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, geçirgen bir zar, 12 Å bir ayırıcı kolu ile kimyasal indirgenebilir çapraz bağlayıcı geçici protein etkileşimleri 1,2,3,4 stabilize etmek için kullanılır. Burada adaptör kompleksi AP-3, 5 endosomes veziküller içine zarı proteinleri tanır ve sıralar çözünebilir bir protein kompleksi ile bu stratejinin bir örneğini verdi . AP-3 seçici çinko taşıyıcı ZnT3 ve lipid kinaz fosfatidilinositol-4-kinaz tip II alfa değil transferrin reseptör 1,4,6 bağlar. Biz...

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		<th>Material Name</th>
		<th>Type</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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<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>
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isolation of labile multi-protein complexes by in vivo controlled cellular cross-linking and immuno-magnetic affinity chromatography

Hücresel makine dinamik doğası, sıklıkla geçici ve / veya zayıf protein dernekler üzerine inşa edilmiştir. Bu düşük afinite etkileşimleri ilgi bir protein etrafında izolasyon ve protein ağlarının belirlenmesi için sıkı yöntemleri engellemektedir. , Kimyasal crosslinkers kullanımı, böylece arıtma için biyokimyasal sınırlamalar atlayarak, labil etkileşimleri seçici istikrar sağlar. Burada bir ayırıcı bir kol ile bir homobifunctional çapraz bağlayıcı kullanır kültür hücreleri için mükellef bir protokol mevcut 12 Å, dithiobis (succinimidyl proprionate) (DSP). DSP molekülü bir disülfit bağ azaltılması bölünmüş. Manyetik boncuklar ile ilgi proteinlerin immünoafinite kromatografisi ile kombine Çapraz bağlama, aksi arıtma dayanamaz protein kompleksleri izolasyonu sağlar. Bu protokol düzenli western blot teknikleri ile uyumlu ve kütle spektrometresi 1 protein tanımlanması için büyütülüyor olabilir. Stephanie A. Zlatic ve Pearl V. Ryder, bu çalışmaya eşit katkıda bulundu.

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

Hücre geçirgen çapraz bağlayıcı DSP [dithiobis-(succinimidyl propiyonat)] stabilize geçici ve labil etkileşimleri<em&gt; In vivo</em&gt; Sıkı protein kompleksi arıtma teknikleri kullanarak kendi izolasyon sağlar. Burada immunoprecipitation protein kompleksleri izolasyonu, kültürde yetişen crosslinking hücreler için kullanılan bir tekniktir.

Dayanıksız Multi-protein Kompleksleri izolasyonu In vivo Kontrollü Hücresel Çapraz Bağlama ve immuno-manyetik Afinite Kromatografisi

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

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Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other ...

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