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

이 작품은 VF에 국립 보건원 (NS42599 및 GM077569)에서 보조금에 의해 지원되었다.

1. Crosslinking 준비 <ol><li> 당신은 표준 튜브 반응 당 단백질 500 μg의 고립을 허용 접시에 세포의 충분한 번호가 필요합니다. 하나의 튜브가 immunoblot으로 관심의 단백질 putative interactors의 식별하기에 충분한 수 있습니다. 이 경우 비드 바운드 자료는 SDS - PAGE 샘플 버퍼 (immunoprecipitation)로 eluted 수 있습니다. 질량 분광법의 분석을 위해, 표준 반응의 숫자는 적어도 10 배 증가되어야하며 단백질 단지는...

<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, 멤브레인 - 침투성, 12의 스페이서 암과 화학적 줄이할 수있는 crosslinker는 과도 단백질 상호 작용 1,2,3,4를 안정화하는 데 사용됩니다. 여기서 우리는 어댑터 복잡한 AP - 3 5 endosomes에서 vesicles에 막 단백질을 인식하고 각종 가용성 단백질 복합이 전략을 exemplified. AP - 3 선택 아연 수송 ZnT3 및 지질 키나제 phosphatidylinositol - 4 - 키나제 타입 II 알파지만 트랜스페린 수용체 1,4,6에</...

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

세포 기계의 동적 특성은 자주 일시적 및 / 또는 약한 단백질 연결에 내장되어 있습니다. 이러한 낮은 친화도 상호 작용 관심의 단백질 주변의 분리 및 단백질 네트워크의 식별에 대한 엄격한 방법을 배제. 화학 crosslinkers의 사용은 따라서 정화에 대한 생화 학적 한계를 거치지 않고 불안 정한 상호 작용의 선택 안정화 수 있습니다. 여기에 우리가의 스페이서 팔로 homobifunctional crosslinker를 사용하는 문화의 세포에 대한 의무 프로토콜을 현재 12, dithiobis - (succinimidyl proprionate) (DSP). DSP는 분자에 존재 disulphide 결합의 감소에 의해 죽습니다. 자석 구슬과 관심의 단백질의 면역 크로마 토그래피와 결합 가교 달리 정화 견딜 않을 단백질 단지의 절연을 수 있습니다. 이 프로토콜은 일반적인 서양 얼룩 기술과 호환되며 그것은 대량 분석법 1 단백질 식별을 위해 확장할 수 있습니다. 스테파니 A. Zlatic와 진주 V. 라이더이 작품에 동일하게 기여.

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

세포 투과 crosslinker DSP [dithiobis - (succinimidyl 프로피 온산)] 일시적 안정화와 불안 정한 상호 작용<em&gt; 생체내에</em엄격한 단백질 복잡한 정화 기술을 사용하여 격리 수 있습니다&gt;. 여기 immunoprecipitation에 의해 단백질 단지의 절연 다음 문화의 성장 crosslinking 전지에 대한 기술을 제시한다.

불안 정한 다중 단백질 단지의 분리에 의해 생체내에 제어 세포 간 연결 및 단백질 자기 동질 크로마 토그래피

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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Biology

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

Photo-Induced Cross-Linking of Unmodified Proteins (PICUP) Applied to Amyloidogenic Peptides

Photo-Induced Cross-Linking of Unmodified Proteins PICUP Applied to Amyloidogenic Peptides

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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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