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 (immünoafinite kromatografisi) tarafından tanınan bir peptid antijeni ile rekabet ile yıkandı, olmalıdır. Bu strateji, immünglobulin protein kompleksleri izolasyonu sağlayacaktır. </li><li> Crosslinking için en uygun hücre yoğunluğu% 75 ve% 90 confluency arasında. Yüksek confluency koşullarda, hücreler, tüm hücrelerin hücre geçirgen çapraz bağlayıcı erişilebilirlik azalır, birbirlerine kazık eğilimindedir. </li><li> Hücre tipi ve deneysel durum hücre plakanın alt tarafında etiketli olmalıdır. Biz rutin olarak tek başına araç ile kontroller içerir. </li><li> DSP çözümü DIŞINDA önce crosslinking tüm çözümler hazırlayın. <ol><li> CaCl 2 0.1 mM CaCl 2 deney başlamadan önce 1 mM (PBS / Ca / Mg) ile fosfat tamponlu salin tampon stok hazırlayın. Ayrıca kalsiyum ve magnezyum iyonlarının hücreleri deney sırasında kültür plakasına yapışması için kritik öneme sahiptir. 4 ° C saklayınız </li><li> 50X Komple Proteaz İnhibitör Kokteyl - 1 ml Milli-Q su içinde çözünmüş 1 tablet. -20 ° C&#39;de muhafaza </li><li> % 20 Triton X-100 10g Triton X -100 dışarı tartılır ve 50 ml Milli-Q Su toplam hacmi içinde sulandırmak. Rock, 4 ° C 4 geceleme ve mağaza ° C kullanana kadar. 1 aydan fazla tutmayın. Bu% 20 hisse senedinin aşağıda açıklanan parçalama ve IP tamponlar hazırlamak için dilüsyonları kullanın. </li><li> PH 7.4 50X DSP Quenching Çözüm 1M TRIS. Oda sıcaklığında saklayın. </li><li> 10x Tampon bir çözüm hazırlayın. 10X Tampon A: <ul><li> 50 mL 1 M Hepes </li><li> 150 mL 5 M NaCl </li><li> 10 ml 0.5 M EGTA </li><li> 250 mcL 2 M MgCl 2 </li><li> PH 7.4 &#39;e ayarlayın </li><li> Son hacim 500 mL getirin </li></ul> 10X Tampon bir çözüm, bir ihtiyaç olarak 1X Buffer seyreltilir. 1x Tampon lizat ve immuno-manyetik yağış tamponlar kullanılır. A 1X Buffer <ul><li> 10 mM HEPES </li><li> 150 mM NaCl </li><li> 1 mM EGTA </li><li> 0.1 mM MgCl 2 </li><li> pH: 7.4 </li></ul></li><li> Lizis Tampon 1X Buffer A +% 0.5 Triton X-100. </li><li> İmmüno-Manyetik Yağış Buffer (IP Tampon) 1X Buffer A +% 0.1 Triton X-100. </li></ol></li></ol> 2. Crosslinking Çözüm hazırlanması <ol><li> DSP çözümü hücrelere başvuran hemen önce hazırlayın. DSP son derece hidrofobik ve PBS / Ca / Mg tampon içine seyreltilmesi önce DMSO içinde çözülmüş olmalıdır. DSP DMSO 1 ml 40 mg çözülür. Bu 100 mM DSP (DSP molekül ağırlığı 404,42 g / mol dür) bir çözüm haline getirir. </li><li> PBS içine DSP / DMSO seyreltme kolaylaştırmak için 37 ° C sipariş PBS / Ca / Mg uygun bir hacmi ısıtmak için. Çeşit çeşit plaka boyutları için gerekli Hacimler: Ortalama 6-plaka 2 mL 10 cm plakası plaka başına 10 ml 15 cm plakası plaka başına 20 ml </li><li> DSP / DMSO sıcak PBS / Ca / Mg her 1 mL stok çözelti 10μL ekleyin. DSP eriyene kadar DSP / DMSO stok solüsyonu damla damla tekrarlanan karıştırma ekleyin. Her 1 mL PBS / Ca / Mg 10 mcL DMSO bir kontrol çözümü olun. </li><li> 10 dakika daha uzun bir buz-su banyosu tüm PBS / Ca / Mg çözümleri yerleştirin. </li></ol> 3. Hücreler Crosslinking için hazırlayın <ol><li> Crosslinking veya araç kontrol inkübasyon için tüm plakaları uyacak bir buz-su banyosu hazırlayın. </li><li> 37 ° C inkübatör plakalarını alın ve hemen buz-su banyosu içine koyun. </li><li> Buz gibi soğuk PBS / Ca / Mg hücreler iki kez yıkayın. (Bkz. bölüm 2.2), çapraz bağlayıcı inkübasyon için kullanacağınız aynı hacimde kullanın. </li><li> Ikinci yıkama çıkarın ve araç kontrolü veya DSP crosslinking tampon çözelti ya ekleyin. </li><li> Iki saat boyunca buz üzerinde inkübe edin. Plakalar tüm hücreleri çözümü ile kaplı olduğundan emin olmak için yaklaşık her yirmi dakikada bir kontrol etmelisiniz. DSP küçük bir miktar çözüm çökelerek görebilirsiniz. Bu normal bir durumdur. </li></ol> 4. DSP Reaksiyon inaktivasyonu <ol><li> PBS / Ca / Mg (her 1 mL PBS / Ca / Mg 20 mcL için 1 M Tris pH: 7.4) 20 mM Tris, pH 7.4 1X DSP soğutmadan çözüm hazırlayın. </li><li> Araç kontrol ve crosslinking çözümler çıkarın. </li><li> Inaktivasyon çözüm buz ekleyin ve 15 dakika boyunca buz üzerinde kuluçkaya yatmaktadır. </li></ol> 5. Hücre Lizis <ol><li> DSP su verme kuluçka sırasında,% 0.5 Triton Tampon X-100 ile komple proteaz inhibitörü kokteyl hücre lizis tamponu hazırlamak. Her 1 mL lizis tamponu için tam 20 mcL 50X stok solüsyonu ekleyin. </li><li> PBS / Ca / Mg, 15 dakika DSP su verme inkübasyon sonra, hücreler iki kez yıkayın. </li><li> 30 dakika lizis tamponu + 4 kaya hücreleri ve Komple ° C ekleyin. Bazı çeşit çeşit plaka boyutları için lizis tamponu hacimleri önerilmektedir: <ul><li> Ortalama 6-plaka 0,5 mL </li><li> 10 cm plakası plaka başına 1 ml </li><li> 15 cm plakası plaka başına 1 ml </li></ul></li><li> Parçalama işleminden sonra, plaka hücreleri toplamak için bir hücre kazıyıcı kullanın. Proteaz aktivitesi en aza indirmek için, buz üzerinde lizatları tutmak için emin olun. </li><li> Spin hücre, en yüksek hızı (15.000 xg) bir tezgah üstü mini-santrifüj 15 dakika 4 ° C lizatları. </li><li> Taze bir tüpe süpernatantı Pipet. Pelet atın. </li><li> Protein düzeyleri analiz ve immunoprecipitation devam. </li></ol> 6. Immuno-manyetik Yağış Boncuk hazırlanması Not: Bu adım, genellikle 2 saat crosslinking inkübasyon doğrudan başından itibaren başlatılır. <ol><li> 30 Dynal immunomagnetic boncuk mcL, 500 mcL IP Buffer (Tampon 1x A +% 0.1 Triton X-100) ve mikrosantrifüj tüpler screwtop uygun miktarda antijen-antikor birleştirin. Negatif kontroller için antikor ücretsiz ve non-spesifik antikor tüpleri hazırlayın. Uygun miktarda antikor ayrı deneyler sırasında bireysel antijenlere göre tespit edilmelidir. Tüm IP tüpler Etiket. </li><li> IP tüpler iki saat boyunca oda sıcaklığında bir sonu aşırı uç rocker inkübe izin verin. Alternatif olarak, crosslinking 4 ° gün önce gece IP tüpler antikor ile inkübe edin. </li><li> Inkübasyondan sonra, tüpün alt kısmında boncuk toplamak için 10 saniyede hızlı bir mini-santrifüj yapın. </li><li> Tüpler tüp alt uzak manyetik boncuk çekin manyetik tutucu, içine kaydırın. Güvenli bir şekilde çıkış yolu boncuklar ile artık kolayca herhangi bir bağlanmamış antikor uzak yıkayabilirsiniz. </li><li> Bir jel yükleme ucu veya kuluçka tampon ve kaldırmak için herhangi bir bağlanmamış antikor P200 ucu gibi küçük bir delik aspiratör ucu kullanın. Inkübasyon hacmi kaldırıldı mümkün olduğu kadar erken IP Buffer 1 ml ekleyin. </li><li> Nazikçe yeniden askıya boncuk, boru kılıfı, ve tüm IP tüpleri üzerinde uç uç rotasyon ile 5 dakika oda sıcaklığında inkübe edin. </li><li> IP Buffer taze bir mililitre ile bir kez daha 5 dakika yıkama adım (6.3 6.6 yoluyla adımları) tekrarlayın. </li><li> Çapraz lizat boncuklar eklenmek üzere hazır olana kadar IP tüpler son yıkama inkübasyon kalır. </li></ol> 7. Immuno-manyetik Boncuk Çapraz Lizat inkübe <ol><li> Son yıkama spin immuno-manyetik bilye sonra, aşağı 10 saniye için bir mini-santrifüj. Sonra manyetik tutucunun içine tüpler kayma devam edin. </li><li> Yine küçük bir delik aspirasyon ucu kullanarak, tüpten kuluçka hacmi çıkarın. Hemen immuno-manyetik bilye içeren tüplere çapraz hücre lizatı (1 mcg / mL lizat varsayarak) 500 mcL ekleyin. Negatif kontrol olarak peptid rekabet olacak, uygun bir miktarda peptit bu noktada dahil edilmelidir. Önceki deneyler, uygun peptid rekabet koşulları belirlemek için yapılmalıdır. Alternatif olarak, bir negatif kontrol olarak lizat ücretsiz immuno-manyetik bilye olmasını isteyebilirsiniz. Bu durumda, hemen immuno-manyetik bilye + Komple Lizat Buffer (Tampon 1x A +% 0.5 Triton X-100) 500 mcL ekleyin. </li><li> Yavaşça boncuk tekrar süspansiyon. Yeniden süspanse boncuk, 2 saat için bir bitiş üzerinde uç dönme 4 ° inkübe edilmelidir. </li></ol> 8. Boncuk Sınırsız Lizat yıkayın <ol><li> Lizat bir mini-santrifüj 10 saniyelik hızlı bir dönüş yeterli inkübasyon süresi etti. Sonra tüpler manyetik tutucu içine kaydırın. Manyetik tutucu deney kalanı için bir buz banyosunda tutulmalıdır. </li><li> Küçük bir delik aspiratör ucu kullanarak, tüm bağlanmamış lizat çıkarın. Tüm bağlanmamış lizat kaldırıldıktan hemen sonra IP Buffer 1 ml ekleyin. </li><li> Tüp kap boncuklar ve yavaşça tekrar süspansiyon haline getirin. </li><li> Boncuk boncuk peletleme adım (8.1) tekrar yeniden süspanse. </li><li> Yine tüm inkübasyon hacmi aspirat ve hemen IP Buffer 1 ml ekleyin, daha sonra tüp kap. </li><li> IP Tampon boncuk yavaşça tekrar süspansiyon. Yeniden süspanse boncuk üzerinde uç uç sallanan ile 4 ° az 5 dakika inkübe edin. </li><li> Boncuk yıkama adımları (8.4) 8.6 üzerinden 4 kez daha tekrarlayın. </li></ol> 9 - Örnek doğasını ve Boncuk toplama <ol><li> Içine tüm immuno-manyetik yağış tüpler yıkanır sonra tekrar tortu boncuk (8.1) ve slayt tüplermanyetik tutucu. </li><li> Immuno-manyetik bilye bağlı proteinler denatüre koşulları kaldırıldı. Aspire son IP Tampon yıkama ve manyetik sahibinden IP tüp kaldırmak. </li><li> 1x Jel Örnek Tampon tekrar tüpün alt kısmında havuz boncuk boncuk pelet Yukarıdaki IP tüp ekleyin. Örnek tampon miktarı kullanılmak üzere jel iyi büyüklüğüne bağlıdır. Jel Örnek Tampon yeniden süspanse boncuk almak için tüpün alt hafifçe dokunmanız gerekebilir. </li><li> Her IP tüpü için (9.3 üzerinden 9.2) denatüre işlemi tekrarlayın. </li><li> Jel Örnek Tampon yeniden süspanse immuno-manyetik bilye sonra 75 ısıtılır ° C denatüre işlemini tamamlamak için 5 dakika. </li><li> Sonra örnek batı blot SDS-PAGE jel üzerinde çalıştırılabilir denatüre olmuştur. Boşaltılmış immuno-Manyetik Boncuk santrifüj yeniden pelet ve manyetik tutucu ile örnek kaldırılır olabilir. </li></ol> 10 Çapraz antijenik Peptitler Kompleksleri (immunoaffiniti Kromatografisi) Elüsyon. <ol><li> Immuno manyetik immunoprecipitation tüplerin sonra manyetik tutucu içine yeniden tortu boncuk (8.1) ve slayt tüpleri, yıkanmış edilmiştir. Süpernatant aspire ve 10 ul Tampon eklemek protein kompleksleri immunoisolation için kullanılan antikor tarafından tanınan antijenik peptit ile desteklenmiştir. 10-200 mcM verimli yürütülmesi için değişen konsantrasyonlarda test etmelidir. </li><li> 4 boncuk 2 saat süreyle inkübe ° C </li><li> Boncuk manyetik stand koyun ve 10 ml yıkandı, malzeme dikkatli bir şekilde kurtarabilirsiniz. Bu süpernatant kaydedin. </li><li> Tampon bir hızla boncuk yıkama ve yıkama atın. Boncuklar kaydedin. </li><li> 1X bir konsantrasyon kaydedilen süpernatant ve boncuklar Jel Örnek Tamponu ekleyin. Bu tüpler 75 ° C&#39;de 5 dakika inkübe edin. </li><li> Boncuk ve peptid eluat SDS-PAGE ile analiz edin. Eluat hem SDS-PAGE leke protein veya immunoblots (Şekil 1B) IgG arındırılmış olmalıdır. IgG Bu malzeme, kütle spektrometresi için uygundur. </li></ol><img src="/files/ftp_upload/1855/1855fig1.jpg" alt="Şekil 1" /> Şekil 1. AP-3 etkileşim protein kompleksleri ve membran proteinlerinin izolasyonu HEK293 hücreleri (A) veya PC12 hücreleri (B) araç kontrol varlığı ya da tedavi (DMSO, tek şerit Şekil 1A) veya DSP (hatta şerit Şekil 1A veya tüm Şekil 1B kulvar). Şekil Aydınlatılmış özleri yalnız boncuklarla (Şekil 1A, Lanes 3-4), transferrin reseptör antikorları (Şekil 1A, Lanes 5-6), ya da AP-3 δ antikorlar (Şekil 1A, Lanes 7-10 ya inkübe edildi. 1B, şerit 1-10). SDS-PAGE örnek tamponu (Şekil 1A) immün kompleksler ile yıkandı, bir başına Tampon veya (Şekil 1B, şerit 1-2), veya δ antijenik peptid amino asitler 680 710 karşılık artan konsantrasyonları ile desteklenmiş Tampon (Şekil 1B, şerit 3-10):;: insan δ-adaptin (1.923.266 gi AAD03777 NCBI). Bu peptid δ antikor bağlar. Peptid elüsyon sonra, süpernatantlar (S) ve boncuklar (B), immunoblotting (Şekil 1B) tarafından analiz edildi. Yanı sıra, clathrin ağır zincir (ÇHC), BLOK-1 altbirim pallidin ve HOPS karmaşık altbirim vps33b: δ, β3 ve σ3 alt birimden karşı antikor tespit AP-3, aşağıdaki çözünür faktörler coprecipitates membran fosfatidilinositol-4-kinaz tip II alfa (PI4KIIα) ve çinko taşıyıcı 3 (ZnT3) proteinleri. Peptid IgG ağır zincir fare yokluğunda Şekil süpernatant yıkandı, unutmayın. 1B.

<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>
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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.

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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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JoVE Journal

Biology

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

17.2K Görüntüleme.  Emory University. Hücre geçirgen çapraz bağlayıcı DSP [dithiobis-(succinimidyl propiyonat)] stabilize geçici ve labil etkileşimleri In vivo Sıkı protein kompleksi arıtma teknikleri kullanarak kendi izolasyon sağlar. Burada immunoprecipitation protein kompleksleri izolasyonu, kültürde yetişen crosslinking hücreler için kullanılan bir tekniktir.

Video: 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

The assembly of amyloidogenic proteins into toxic oligomers is a seminal event in the pathogenesis of protein misfolding diseases, including Alzheimer's, Parkinson's, and Huntington's diseases, hereditary amyotrophic lateral sclerosis, and type 2 diabetes. Owing to the metastable nature of these protein assemblies, it is difficult to assess their oligomer size distribution quantitatively using classical methods, such as electrophoresis, chromatography, fluorescence, or dynamic light scattering. Oligomers of amyloidogenic proteins exist as metastable mixtures, in which the oligomers dissociate into monomers and associate into larger assemblies simultaneously. PICUP stabilizes oligomer populations by covalent cross-linking and when combined with fractionation methods, such as sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) or size-exclusion chromatography (SEC), PICUP provides snapshots of the oligomer size distributions that existed before cross-linking. Hence, PICUP enables visualization and quantitative analysis of metastable protein populations and can be used to monitor assembly and decipher relationships between sequence modifications and oligomerization1. Mechanistically, PICUP involves photo-oxidation of Ru2+ in a tris(bipyridyl)Ru(II) complex (RuBpy) to Ru3+ by irradiation with visible light in the presence of an electron acceptor. Ru3+ is a strong one-electron oxidizer capable of abstracting an electron from a neighboring protein molecule, generating a protein radical1,2. Radicals are unstable, highly-reactive species and therefore disappear rapidly through a variety of intra- and intermolecular reactions. A radical may utilize the high energy of an unpaired electron to react with another protein monomer forming a dimeric radical, which subsequently loses a hydrogen atom and forms a stable, covalently-linked dimer. The dimer may then react further through a similar mechanism with monomers or other dimers to form higher-order oligomers. Advantages of PICUP relative to other photo- or chemical cross-linking methods3,4 include short (&#8804;1 s) exposure to non-destructive visible light, no need for pre facto modification of the native sequence, and zero-length covalent cross-linking. In addition, PICUP enables cross-linking of proteins within wide pH and temperature ranges, including physiologic parameters. Here, we demonstrate application of PICUP to cross-linking of three amyloidogenic proteins the 40- and 42-residue amyloid &beta;-protein variants (A&beta;40 and A&beta;42), and calcitonin, and a control protein, growth-hormone releasing factor (GRF).

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

Photo-induced cross-linking of unmodified proteins (PICUP) allows characterization of oligomer size distribution in metastable protein mixtures. We demonstrate application of PICUP to three representative amyloidogenic peptides the 40- and 42-residue forms of amyloid &beta;-protein, and calcitonin, and a control peptide growth-hormone releasing factor.

Fotoğraf Bağlı Çapraz Bağlanma Değiştirilmemiş Proteinler (PICUP) amiloidojenik Peptitler Uygulanan

The assembly of amyloidogenic proteins into toxic oligomers is a seminal event in the pathogenesis of protein misfolding diseases, including Alzheimer's, ...

Biyoloji

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.

S. cerevisiae, kantitatif proteomik protein kompleksleri tanımlanması

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as ...

Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy

Full insight into the mechanisms of living cells can be achieved only by investigating the key processes that elicit and direct events at a cellular level. To date the shear complexity of biological systems has caused precise single-molecule experimentation to be far too demanding, instead focusing on studies of single systems using relatively crude bulk ensemble-average measurements. However, many important processes occur in the living cell at the level of just one or a few molecules; ensemble measurements generally mask the stochastic and heterogeneous nature of these events. Here, using advanced optical microscopy and analytical image analysis tools we demonstrate how to monitor proteins within a single living bacterial cell to a precision of single molecules and how we can observe dynamics within molecular complexes in functioning biological machines. The techniques are directly relevant physiologically. They are minimally-perturbative and non-invasive to the biological sample under study and are fully attuned for investigations in living material, features not readily available to other single-molecule approaches of biophysics. In addition, the biological specimens studied all produce fluorescently-tagged protein at levels which are almost identical to the unmodified cell strains ("genomic encoding"), as opposed to the more common but less ideal approach for generating significantly more protein than would occur naturally ('plasmid expression'). Thus, the actual biological samples which will be investigated are significantly closer to the natural organisms, and therefore the observations more relevant to real physiological processes.

Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy

Here we demonstrate the protocols for performing single-molecule fluorescence microscopy on living bacterial cells to enable functional molecular complexes to be detected, tracked and quantified.

Tek Moleküler Kompleksleri görselleştirme In Vivo Gelişmiş Floresan Mikroskopi

Full insight into the mechanisms of living cells can be achieved only by investigating the key processes that elicit and direct events at a cellular ...

ELIME (Enzyme Linked Immuno Magnetic Electrochemical) Method for Mycotoxin Detection

Immunoassays are a valid alternative to the more expensive and time consuming quantitative HPLC or GC1, 2 methods for the screening detection of hazardous mycotoxins in food commodities. In this protocol we show how to fabricate and interrogate an electrochemical competitive Enzyme linked immunomagnetic assay based on the use of magnetic beads as solid support for the immunochemical chain3 and screen printed electrodes as sensing platform.
Our method aims to determine the total amount of HT-2 and T-2 toxins, mycotoxins belonging to the trichothecenes family and of great concern for human health4. The use of an antibody clone with a cross reactivity of 100% towards HT-2 and T-2 allows to simultaneously detect both toxins with similar sensitivity5.
The first step of our assay is the coating step where we immobilize HT2-KLH conjugate toxin on the surface of magnetic beads. After a blocking step, necessary to avoid non-specific absorptions, the addition of a monoclonal antibody allows the competition between immobilized HT-2 and free HT-2 or T-2 present in the sample or dissolved in a standard solution.
At the end of the competition step, the amount of monoclonal antibody linked to the immobilized HT-2 will be inversely proportional to the amount of toxin in the sample solution.
A secondary antibody labeled with alkaline phosphatase (AP) is used to reveal the binding between the specific antibody and the immobilized HT-2. The final measurement step is performed by dropping an aliquot of magnetic bead suspension, corresponding to a specific sample/standard solution, on the surface of a screen-printed working electrode; magnetic beads are immobilized and concentrated by means of a magnet placed precisely under the screen-printed electrode. After two minutes of incubation between magnetic beads and a substrate for AP, the enzymatic product is detected by Differential Pulse Voltammetry (DPV) using a portable instrument (PalmSens) also able to initiate automatically eight measurements within an interval of few seconds.

ELIME Enzyme Linked Immuno Magnetic Electrochemical Method for Mycotoxin Detection

A protocol to detect trichothecenes (mycotoxins of concern for human health) using a newly developed screening method based on a competitive immunochemical method and a final electrochemical detection is demonstrated.

Mikotoksin Algılama ELIME (Enzim İmmüno Manyetik Elektrokimyasal Bağlantılı) Yöntem

Immunoassays are a valid alternative to the more expensive and time consuming quantitative HPLC or GC1, 2 methods for the screening detection of hazardous ...

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal lines have enabled biological processes to be studied in the context of a living, and in some instances even behaving, organism. In this protocol we will describe how to anesthetize intact Drosophila larvae, using the volatile anesthetic desflurane, to follow the development and plasticity of synaptic populations at sub-cellular resolution1-3. While other useful methods to anesthetize Drosophila melanogaster larvae have been previously described4,5,6,7,8, the protocol presented herein demonstrates significant improvements due to the following combined key features: (1) A very high degree of anesthetization; even the heart beat is arrested allowing for lateral resolution of up to 150 nm1, (2) a high survival rate of &gt; 90% per anesthetization cycle, permitting the recording of more than five time-points over a period of hours to days2 and (3) a high sensitivity enabling us in 2 instances to study the dynamics of proteins expressed at physiological levels. In detail, we were able to visualize the postsynaptic glutamate receptor subunit GluR-IIA expressed via the endogenous promoter1 in stable transgenic lines and the exon trap line FasII-GFP1. (4) In contrast to other methods4,7 the larvae can be imaged not only alive, but also intact (i.e. non-dissected) allowing observation to occur over a number of days1. The accompanying video details the function of individual parts of the in vivo imaging chamber2,3, the correct mounting of the larvae, the anesthetization procedure, how to re-identify specific positions within a larva and the safe removal of the larvae from the imaging chamber.

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

This protocol describes a reliable method for anesthetization and imaging of intact Drosophila melanogaster larvae. We have utilized the volatile anesthetic desflurane to allow for repetitive imaging at sub-cellular resolution and re-identification of structures for up to a few days1.

Alt hücresel Çözünürlük bozulmamış Drosophila Larva in vivo Görüntüleme

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal ...

Isothermal Titration Calorimetry for Measuring Macromolecule-Ligand Affinity

Isothermal titration calorimetry (ITC) is a useful tool for understanding the complete thermodynamic picture of a binding reaction. In biological sciences, macromolecular interactions are essential in understanding the machinery of the cell. Experimental conditions, such as buffer and temperature, can be tailored to the particular binding system being studied. However, careful planning is needed since certain ligand and macromolecule concentration ranges are necessary to obtain useful data. Concentrations of the macromolecule and ligand need to be accurately determined for reliable results. Care also needs to be taken when preparing the samples as impurities can significantly affect the experiment. When ITC experiments, along with controls, are performed properly, useful binding information, such as the stoichiometry, affinity and enthalpy, are obtained. By running additional experiments under different buffer or temperature conditions, more detailed information can be obtained about the system. A protocol for the basic setup of an ITC experiment is given.

A general protocol for the use of isothermal titration calorimetry to monitor the binding thermodynamics for biological systems with moderate binding affinities is presented.

Makromolekül-ligand Affinity Ölçüm İzotermal Titrasyon Kalorimetre

Isothermal titration calorimetry (ITC) is a useful tool for understanding the complete thermodynamic picture of a binding reaction.  In biological ...

Chromatin Isolation by RNA Purification (ChIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression 1,2,3,4,5,6,7. The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion 8,9. While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation 10,11. However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide 3,12,13, but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex 14; HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex 13.
Prior studies mapping RNA occupancy at chromatin have revealed substantial insights 15,16, but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution 17. This method, Chromatin Isolation by RNA Purification (ChIRP) (Figure 1), is based on affinity capture of target lncRNA:chromatin complex by tiling antisense-oligos, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. ChIRP is applicable to many lncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA's structure or functional domains.

Chromatin Isolation by RNA Purification ChIRP

ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.

RNA Saflaştırma tarafından Kromatin İzolasyon (chirp)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems1, 2. Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria1-6. In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes1, 2, 7. Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast8, 9, and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system10. Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large-scale cultures. Tandem mass spectrometry is then used to identify the stably co-purifying proteins with high sensitivity (low nanogram detection limits).
Here, we describe detailed step-by-step procedures we commonly use for systematic protein tagging, purification and mass spectrometry-based analysis of soluble protein complexes from E. coli, which can be scaled up and potentially tailored to other bacterial species, including certain opportunistic pathogens that are amenable to recombineering. The resulting physical interactions can often reveal interesting unexpected components and connections suggesting novel mechanistic links. Integration of the PPI data with alternate molecular association data such as genetic (gene-gene) interactions and genomic-context (GC) predictions can facilitate elucidation of the global molecular organization of multi-protein complexes within biological pathways. The networks generated for E. coli can be used to gain insight into the functional architecture of orthologous gene products in other microbes for which functional annotations are currently lacking.

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.

Protein Kompleksi belirlenmesi<em&gt; Escherichia coli</em&gt; Tandem Kütle Spektrometresi ile Kombine Sıralı Peptid Affinity Arıtma kullanarak

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the ...

AFM-based Mapping of the Elastic Properties of Cell Walls: at Tissue, Cellular, and Subcellular Resolutions

We describe a recently developed method to measure mechanical properties of the surfaces of plant tissues using atomic force microscopy (AFM) micro/nano-indentations, for a JPK&#160;AFM. Specifically, in this protocol we measure the apparent Young&#8217;s modulus of cell walls at subcellular resolutions across regions of up to 100 &#181;m&#160;x 100 &#181;m in floral meristems, hypocotyls, and roots. This requires careful preparation of the sample, the correct selection of micro-indenters and indentation depths. To account for cell wall properties only, measurements are performed in highly concentrated solutions of mannitol in order to plasmolyze the cells and thus remove the contribution of cell turgor pressure.
In contrast to other extant techniques, by using different indenters and indentation depths, this method allows simultaneous multiscale measurements, i.e. at subcellular resolutions and across hundreds of cells comprising a tissue. This means that it is now possible&#160;to spatially-temporally characterize the changes that take place in the mechanical properties of cell walls during development, enabling these changes to be correlated with growth and differentiation. This represents a key step to understand how coordinated microscopic cellular changes bring about macroscopic morphogenetic events.
However, several limitations remain: the method can only be used on fairly small samples (around 100 &#181;m in diameter) and only on external tissues; the method is sensitive to tissue topography; it measures only certain aspects of the tissue&#8217;s complex mechanical properties. The technique is being developed rapidly and it is likely that most of these limitations will be resolved in the near future.

We describe a method to map mechanical properties of plant tissues using an atomic force microscope (AFM). We focus on how to record mechanical changes that take place in cell walls during plant development at wide-field mesoscale, enabling these changes to be correlated with growth and morphogenesis.

Hücre Duvarları Elastik Özelliklerinin AFM-tabanlı Haritalama: doku, hücresel ve Subselüler Çözünürlüklerde

We describe a recently developed method to measure mechanical properties of the surfaces of plant tissues using atomic force microscopy (AFM) ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

Yetişkin Fare Kalvaryal Kemik İliği içinde Hematopoietik kök hücrelerin Vivo 4-Boyutlu Takip

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...