Bu eser için bir arkadaş grubu tarafından desteklenen S.H.-A. NIH/NCATS (TL1TR001104), Welch Vakfı Hibe (I-1827) ve NIGMS (RGM120502A) Z.W. Z.W. bir R01 hibe'tıbbi araştırma ve kanser önleme ve Texas akademik ve Araştırma Enstitüsü (R1222) Virginia Murchison Linthicum uzman olduğunu.

1. hazırlayın örnekleri
<ol><li>Kültür 2 x 106 amiloid HT-29 kolon kanser hücrelerinin 10 ml, Dulbecco'nın modifiye kartal orta (% 10 fetal sığır serum ve penisilin-streptomisin içeren DMEM) 10 cm doku kültürü tabak içinde üreten. Kültür hücreleri 37 ° C kuluçka %5 CO2gece.<ol>
<li>Sonra % 80 confluency için hücrelerin büyümesine, fosfat tamponlu tuz (PBS) 10 mL hücrelerle yıka. 3 mL tripsin çözeltisi ekleyin ve 3 dk 37 ° C'de kuluçkaya.</li>
		<li>Hücreleri yemek tamamen Disosiye sonra kültür orta 10 mL ekleyin ve 15 mL konik tüp 10 mL pipet hücrelerle aktarmak. Senaryo Özeti 3 dakika oda sıcaklığında 1000 x g santrifüj kapasitesi. Orta Aspire edin, 5 mL kültür ortamının hücrelerde resuspend ve bir hücre sayaç kullanarak hücreleri saymak. Her iki 10 cm tabak tabak 2 x 106 hücreler.</li>
		<li>Hücreleri uygun ve gecede 37 ° C kuluçka %5 CO2kurtarmak için izin verir. Amyloids 20 ng/mL tümör nekrozis faktör-alfa (TNF-α), 100 nM şaplak atmak mimetic ve 20 µM pan-caspase inhibitörü Z-VAD-FMK11ile oluşumu ikna etmek için bir yemek için tedavi uygulanır. Kasanın TSZ kısaltılır. Araç ile diğer çanak bir denetim olarak tedavi.</li>
	</ol>
</li>
	<li>Uygun süreyi sonra genellikle 6 h, lysate hücre hasat.<ol>
<li>Plastik bir sıyırıcı plaka kapalı hücre scrape ve 10 mL pipet 15 mL konik tüp içine aktarmak için kullanın. 1000 x g 4 ° C'de 3 dk de hücreleri santrifüj kapasitesi</li>
		<li>Hücreleri tarafından 10 mL buz soğuk PBS resuspending ve 1000 x g 4 ° C'de 3 dk de centrifuging 2 kez yıkamak PBS çözüm Aspire edin. Not: İşlemi burada hücre Pelet sıvı azot içinde dondurma ve ˗80 ° C ilâ 1 aylığına depolayarak duraklatılmış.</li>
		<li>Hücre Pelet 1.5 mL microcentrifuge tüp aktarmak ve buz üzerinde 30 dk için lizis arabellek 0.3 ml kuluçkaya. 4 ° C'de 15 dakika 20.000 x g, santrifüj Süpernatant lysate tüm cep telefonu var. Not: İşlemi burada örnek-20 ° C'de için birkaç ay kadar depolayarak duraklatılmış.</li>
		<li>Bir Bradford tahlil üreticisinin protokolüne göre tarafından protein konsantrasyonu ölçmek. 4 x 3 µg/µL örnek 20 µL hazırlamak ve 10 dakika oda sıcaklığında kuluçkaya SDD-yaş yükleme arabellek ekleyin.</li>
	</ol>
</li>
</ol>2. hazırlayın ve jelleri çalıştırın
<ol><li>200 ml 1 x Tris-asetat arabelleği (TAE) bir cam kabı ve özel eritmek için bir mikrodalga Isı özel 2 g ekleyin. 1 mL % 20 SDS % 0,1 SDS son bir konsantrasyon için ekleyin. Karıştırmak için dikkatli bir şekilde girdap. Kabarcıklar SDS eklenmesinden sonra oluşturmamanız için dikkat ediniz.</li>
	<li>Özel çözüm 15 cm x 14 cm jel levha dökün. 1-mL pipet herhangi bir kabarcıkları ortadan kaldırmak için kullanın. Bir 20-iyi tarak en üste yerleştirmek.</li>
	<li>İlk Boyut: pipet 60 µg, her hücrenin sağ şeritte lysate. (Boya açık yaklaşık ¾ jel yoluyla olana) jel 60 V 4 h için çalıştırın çalışan tampon olarak % 0,1 SDS içeren TAE kullanarak.</li>
	<li>İkinci Boyut: jel 90 ° saat yönünün tersine (Şekil 2A) dikkatle döndürün. Jel 60 V 4 h için çalıştırın. Not: Genel çalışan 4 V/cm jel uzunluğu bir durumdur. Çalışan koşulların tam olarak birinci ve ikinci boyutları için aynı olduğunu önemlidir.</li>
</ol>3. transfer
<ol><li>Proteinler polivinilidin difluoride (PVDF) membran (Şekil 2B) aktarmak için kılcal aktarma yöntemini kullanın.<ol>
<li>Aktarma arabelleği için 500 mL ekleyin bir yaklaşık 20 cm x 20 cm kapsayıcı. 5-cm yüksek yığın kağıt havlu konteyner yanında yapmak. Yığının üst yüzey alanı jel boyutları büyük olması gerekir.</li>
		<li>14 cm x 15 cm filtre kağıdı Aktarım arabellek emmek ve kağıt havlu yığını üzerine yerleştirin. Hiçbir kırışıklık veya kabarcıklar ile kağıda yerleştirmek için dikkat ediniz. Filtre kağıt başka bir parça ile tekrar.</li>
		<li>14 cm x 15 cm PVDF membran için 30 metanol içinde etkinleştirmek s sonra filtre tüm kabarcıklar kaldırmak için bir rulo kullanmak için dikkat çekici kağıt üzerinde katman. Jel Aktarım arabellek ile durulayın ve yine herhangi bir kabarcıklar inişli çıkışlı membran üstüne katman.</li>
		<li>Plastik film ile yakın Aktarım arabellek kapsayıcıya kağıt havlu kenarı kapsar. Bir sonraki adımda inşa kağıt köprü kağıt havlu yığını ile doğrudan temas gelebilir değil önemlidir.</li>
		<li>15 cm x 35 cm filtre kağıt Aktarım arabellek emmek ve böylece bir ucunu üst jel kapsar ve diğer ucunu Aktarım arabellek kapsayıcısında yerleştirin. Başka bir köprü ile yineleyin.</li>
		<li>Kapsayıcı plastik wrap ile kapak ve gecede oda sıcaklığında bekletin.</li>
	</ol>
</li>
</ol>4. Western Blot algılama
<ol><li>Durulama zar 50 mL % 0.05 içeren PBS ile ara-20 (PBST) olarak tehlikeli madde kabına 20 cm × 20 cm. 20 mL % 5 süt PBST içinde ekleyin. Rock engellemek 30 dakika oda sıcaklığında.</li>
	<li>Tavşan anti-MLKL antikor PBST sütte %5 20 mL içine 10 µL pipet ve kapsayıcıya ekle. Gecede 4 ° C'de rocker üzerinde kuluçkaya</li>
	<li>Membran PBST 20 mL için 5 dk. tekrar 5 kere yıkayın.</li>
	<li>Anti-rabbit-HRP 20 ml % 5 süt PBST içinde 4 µL pipet ve kapsayıcıya ekle. 2 h için oda sıcaklığında rocker üzerinde kuluçkaya.</li>
	<li>Membran PBST 20 mL için 5 dk. tekrar 5 kere yıkayın.</li>
	<li>Gelişmiş Kemiluminesan substrat (ECL) ekleyin ve röntgen filmleri üreticinin protokolüne göre için maruz.</li>
</ol>

<ol>
	<li>Ross, C. A., Poirier, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+aggregation+and+neurodegenerative+disease.">Protein aggregation and neurodegenerative disease.</a> Nature medicine. 10, 10-17 (2004).</li><li>Hou, F., 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=MAVS+forms+functional+prion-like+aggregates+to+activate+and+propagate+antiviral+innate+immune+response.">MAVS forms functional prion-like aggregates to activate and propagate antiviral innate immune response.</a> Cell. 146, 448-461 (2011).</li><li>Li, 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=The+RIP1/RIP3+necrosome+forms+a+functional+amyloid+signaling+complex+required+for+programmed+necrosis.">The RIP1/RIP3 necrosome forms a functional amyloid signaling complex required for programmed necrosis.</a> Cell. 150, 339-350 (2012).</li><li>Liu, 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=MLKL+forms+disulfide+bond-dependent+amyloid-like+polymers+to+induce+necroptosis.">MLKL forms disulfide bond-dependent amyloid-like polymers to induce necroptosis.</a> Proceedings of the National Academy of Sciences of the United States of America. 114, 7450-7459 (2017).</li><li>Uptain, S. M., Lindquist, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prions+as+protein-based+genetic+elements.">Prions as protein-based genetic elements.</a> Annual review of microbiology. 56, 703-741 (2002).</li><li>Halfmann, R., Lindquist, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+for+amyloid+aggregation+by+Semi-Denaturing+Detergent-Agarose+Gel+Electrophoresis.">Screening for amyloid aggregation by Semi-Denaturing Detergent-Agarose Gel Electrophoresis.</a> J Vis Exp. , (2008).</li><li>Sipe, J. 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=Nomenclature+2014:+Amyloid+fibril+proteins+and+clinical+classification+of+the+amyloidosis.">Nomenclature 2014: Amyloid fibril proteins and clinical classification of the amyloidosis.</a> Amyloid : the international journal of experimental and clinical investigation : the official journal of the International Society of Amyloidosis. 21, 221-224 (2014).</li><li>Eisenberg, D. S., Sawaya, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+Studies+of+Amyloid+Proteins+at+the+Molecular+Level.">Structural Studies of Amyloid Proteins at the Molecular Level.</a> Annual review of biochemistry. 86, 69-95 (2017).</li><li>Bagriantsev, S. N., Kushnirov, V. V., Liebman, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in Enzymology. 412, 33-48 (2006).</li><li>Sun, L., 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=Mixed+lineage+kinase+domain-like+protein+mediates+necrosis+signaling+downstream+of+RIP3+kinase.">Mixed lineage kinase domain-like protein mediates necrosis signaling downstream of RIP3 kinase.</a> Cell. 148, 213-227 (2012).</li><li>He, 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=Receptor+interacting+protein+kinase-3+determines+cellular+necrotic+response+to+TNF-alpha.">Receptor interacting protein kinase-3 determines cellular necrotic response to TNF-alpha.</a> Cell. 137, 1100-1111 (2009).</li></ol>

Yazarlar hiçbir çıkar çatışması beyan ederim.

En önemli bir özelliği, 2D SDD-yaş Elektroforez koşullar birinci ve ikinci boyutu için aynı olmasıdır. Benzer bir şekilde her iki sırasında geçiş lifleri 45 ° (hiçbir ayrılma veya bozulma oluştuğunu gösteren), keskin bir çapraz çizgi bağlıdır algılama yeteneğini electrophoreses. Farklı koşullar, örneğin voltaj veya çalışma süreyi değiştirme kullanarak bu sonuçlar karanlık olacak. Geleneksel 1 D için olduğu gibi bu amiloid ve amiloid benzeri lifleri bozabilir olacaktır Ayrıca, SD...

Protein misfolding nedeniyle amiloid lifleri oluşumu, Alzheimer hastalığı, Parkinson hastalığı ve Huntington hastalığı1gibi patolojik koşullarda bir rol oynamaya uzun bilinmektedir. Daha yakın zamanlarda, amiloid veya amiloid benzeri liflerin oluşumu sinyal yolları insanlar Anti-yayılmacı doğuştan gelen bağışıklık yanıtı2 necroptosis3,ve4sırasında dahil olmak üzere ve daha düşük organizmalar bir parçası olmak için gösterilen Maya5,6gibi. Bu nedenle, bu elyaf laboratuarda algılama yeteneğini önemlidir. Şu anda, amiloid ve amiloid benzeri lifler algılamak için üç ana yolu vardır: Boyalar, EM ve SDD-yaş kullanımı.
Boya, Kongo kırmızısı veya Thioflavin T, gibi kullanımı hızlı ve kolayca detectable mikroskopi veya spektroskopisi7kullanarak olmanın avantajı sunuyor. Ancak, mikroskopi, Kongo kırmızısı, söz konusu olduğunda tarafından hangi proteinleri oluşturan lifler veya boyutu lifler, hiçbir özgüllük algılar. Benzer şekilde, Kongo kırmızısı veya Thioflavin T bağlayıcı protein kompleksleri için algılamak için spektroskopisi kullanımı yalnızca bir pozitif veya negatif sonuç sağlar.
EM lifleri ve aynı zamanda lif uzunluğu ve çapı8hakkında kantitatif bilgi varlığının kesin kanıt sağlar. Ancak, bu yöntem çok sıkı arıtma gerektirir. Ayrıca, EM pahalı ekipman kullanan bir özel bir tekniktir.
SDD-yaş SDS dayanıklı mega Dalton protein kompleksleri amiloid veya amiloid benzeri lifler de dahil olmak üzere tespit etmek için kullanılmıştır. Birçok avantaj sunar. İlk olarak, arıtma liflerinin gerektirmez ve peform9için kolaydır. İkinci olarak, göreli boyutu ve fiber heterogenicity miktarı dahil olmak üzere liflerinin boyutu hakkında Nitel bilgi sağlar. Western Blot Elektroforez sonra gerçekleştirilen çünkü son olarak, bu SDD-yaş yarı denaturing olduğu için bazı epitopları hangi gizli kalmasını unutulmamalıdır, ancak kendisi için herhangi bir proteinin bir antikor olduğunu var olup olmadığını belirlemek kolaydır algılama antikor tarafından olmak zordur.
Son zamanlarda, reseptör etkileşen protein kinaz 1 (RIPK1) ve 3 (RIPK3) bildirilmiştir formu amiloid lifleri için platformlar necroptosis sırasında nekroz3programlanmış bir tür sinyal olarak hizmet etmek için. Bu iplikler okurken, bizim laboratuvar lineage kinaz etki alanı gibi (MLKL), karışık başka bir necroptosis ilişkili protein de amiloid benzeri lifler4kurdu gösterdi. Ancak, SDD-yaş ile incelenmesi üzerine, MLKL lifleri boyutunu birbirlerine (şekil 1) aynı çıktı RIPK1 ve RIPK3 lifler farklı çıktı. MLKL necroptosis necrosome10denilen karmaşık sinyal oluşturmak için RIPK1/RIPK3 için bağlar tanınmış olduğundan bu beklenmedik bir şeydi.
En az iki açıklaması vardır. İlk olarak, iki tamamen farklı amiloid benzeri lifler necroptosis sırasında bir içeren RIPK1/RIPK3 ve diğer içeren MLKL şeklinde. İkinci olarak, tek bir içeren RIPK1/RIPK3/MLKL necroptosis ama MLKL ilişkilendirmesiyle diğer proteinler sırasında oluşmuş amiloid benzeri lifler zayıf yeterince SDD-yaş sırasında ayrışıp türüdür.
Bu sorunu çözmek için bir iki boyutlu (2D) SDD yaşından öneriyorum. SDD-yaş-kararlı amiloid veya amiloid benzeri lifler aynı geçiş desen birinci ve ikinci boyut Elektroforez sırasında olacaktır. Bu tespit bir membran proteinleri aktarma ve Western blot taşıma sonra olacak. İstikrarlı elyaf keskin köşegen desen sergileyecek. Bu herhangi bir sapma lifler SDS-Elektroforez nedeniyle değişiklikleri meydana öneririz.

<table><tbody><tr><td>gel electrophoresis unit</td><td>Fisher</td><td>HE99XPRO</td><td>appratus for gel running.</td></tr><tr><td>agrose</td><td>VWR</td><td>97062-250</td><td>For agarose gel.</td></tr><tr><td>paper tower</td><td>Fisher</td><td>19-120-2484</td><td>for transfering</td></tr><tr><td>filter paper</td><td>VWR</td><td>21427-386</td><td>for transfering</td></tr><tr><td>PVDF membrane</td><td>Bio-Rad</td><td>162-0177</td><td>for transfering</td></tr><tr><td>blocking milk powder</td><td>Bio-Rad</td><td>1706404XTU</td><td>for blocking in Western blot</td></tr><tr><td>MLKL antibody</td><td>Genetex</td><td>GTX107538</td><td>rabbit anti-MLKL antibody</td></tr><tr><td>RIPK1 antibody</td><td>BD Biosciences</td><td>610459</td><td>mouse anti-RIPK1 antibody</td></tr><tr><td>RIPK3 antibody</td><td>gift from Dr. Xiaodong Wang</td><td></td><td>rabbit anti-RIPK3. See refenence 11.</td></tr><tr><td>anti-Rabbit-HRP</td><td>Sigma</td><td>A6154</td><td>secondary antibody</td></tr><tr><td>ECL</td><td>Fisher</td><td>WBKLS0500</td><td>Western chemiluminescent HRP substrate</td></tr><tr><td>X-ray film</td><td>Fisher</td><td>F-BX810</td><td>for Western blot result</td></tr><tr><td>DMEM</td><td>Sigma</td><td>D6429</td><td>for cell culture</td></tr><tr><td>fetal bovine serum</td><td>Sigma</td><td>F4135</td><td>for cell culture</td></tr><tr><td>penicilin-streptomycin</td><td>Sigma</td><td>P4333</td><td>for cell culture</td></tr><tr><td>Trypsin solution</td><td>Sigma</td><td>T4049</td><td>for cell culture</td></tr><tr><td>PBS for tissue culture</td><td>Sigma</td><td>D8662</td><td>for cell culture</td></tr><tr><td>recombinant TNF</td><td>made in our lab</td><td></td><td>for inducing necroptosis. See reference 11.</td></tr><tr><td>smac-mimetic</td><td>gift from Dr. Xiaodong Wang</td><td></td><td>for inducing necroptosis. See reference 11.</td></tr><tr><td>ZVAD-FMK</td><td>ApexBio</td><td>A1902</td><td>for inducing necroptosis. See reference 11.</td></tr><tr><td>Cell Counter</td><td>Bio-Rad</td><td>1450102</td><td>Model TC20; for counting cells</td></tr><tr><td>Pierce&amp;trade; BCA Protein Assay Kit</td><td>Thermo Scientific</td><td>23225</td><td>for measuring protein concentration in cell lysates</td></tr><tr><td>Cell lifter</td><td>Fisher</td><td>07-200-364</td><td>to remove cells from dish</td></tr><tr><td>Lysis Buffer (1 L)</td><td></td><td></td><td>20 mL 1 M Tris pH 7.4&lt;br/&gt; 10 mL glycerol&lt;br/&gt; 30 mL 5 M NaCl&lt;br/&gt; 840 mL ddH&lt;sub&gt;2&lt;/sub&gt;O&lt;br/&gt; 10 mL Triton-X100&lt;br/&gt; (protease and phosphates inhibitors as desired)</td></tr><tr><td>10X TAE (1 L)</td><td></td><td></td><td>48.4 g Tris base&lt;br/&gt; 11.42 mL glacial acetic acid&lt;br/&gt; 20 mL 0.5M EDTA pH 8&lt;br/&gt; ddH&lt;sub&gt;2&lt;/sub&gt;0 to 1 L</td></tr><tr><td>4X SDD-AGE loading buffer (50 mL)</td><td></td><td></td><td>5 mL 10X TAE&lt;br/&gt; 10 mL glycerol&lt;br/&gt; 4 mL 20% SDS&lt;br/&gt; 0.5 mL 10% bromophenol blue&lt;br/&gt; 31 mL ddH&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>TBS Transfer Buffer (1 L)</td><td></td><td></td><td>20 mL 1 M Tris pH 7.4&lt;br/&gt; 30 mL 5 M NaCl&lt;br/&gt; 950 mL ddH&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>PBST Wash Buffer (1 L)</td><td></td><td></td><td>100 mL 10xPBS&lt;br/&gt; 800 mL ddH&lt;sub&gt;2&lt;/sub&gt;O&lt;br/&gt; 1 mL Tween20</td></tr><tr><td>10X PBS (10 L)</td><td></td><td></td><td>800 g NaCl&lt;br/&gt; 20 g KCl&lt;br/&gt; 144 g Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;&amp;middot;2H&lt;sub&gt;2&lt;/sub&gt;O&lt;br/&gt; 24 g KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;&lt;br/&gt; add ddH&lt;sub&gt;2&lt;/sub&gt;O to 10 L</td></tr></tbody></table>

use of two dimensional semi-denaturing detergent agarose gel electrophoresis to confirm size heterogeneity of amyloid or amyloid-like fibers

Amiloid Dil veya amiloid benzeri lifler birçok insan hastalıkları ile ilişkili olan ve şimdi birçok sinyal yolları için önemli olduğunu keşfetti edilmektedir. Kolayca bu elyaf çeşitli deneysel koşullarda oluşumu algılama yeteneğini potansiyel işlevleri anlamak için önemlidir. Pek çok yöntem lifler, algılamak için kullanılan ama bazı dezavantajları olmadan olmaz. Örneğin, elektron mikroskobu (EM) veya Kongo kırmızısı veya Thioflavin T ile sık sık boyama arıtma liflerinin gerektirir. Öte yandan, yarı denaturing deterjan özel Jel Elektroforez (SDD-yaş) hücre özleri arıtma olmadan SDS dayanıklı amiloid benzeri lifler algılanmasını sağlar. Buna ek olarak, boyut farkı liflerinin karşılaştırmasını sağlar. Daha da önemlisi, Western Blot tarafından spesifik proteinlerin lifler içinde tanımlamak için kullanılabilir. Daha az zaman alıcı ve labs daha geniş bir dizi için daha kolay erişilebilir. SDD-yaş sonuçları genellikle değişken heterojenite derecesini gösterir. Protein kompleksi SDS huzurunda Elektroforez sırasında ayrılma heterojenite parçası oluşur olup olmadığını sorusunu gündeme getiriyor. Bu nedenle, ikinci bir boyut SDD-ÇAĞINDA görülen boyutu heterojenite gerçekten fiber heterojenite vivo içinde bir sonucu ve bozulma ya da ayrılma sırasında proteinlerin bazı değil bir sonucu olup olmadığını belirlemek için SDD-Age istihdam Elektroforez. Bu yöntem, amiloid veya amiloid benzeri liflerin kısmen SDD-yaş işlemi sırasında dissociating değil hızlı, nitel onay sağlar.

Sonra necroptosis indüksiyon, RIPK1 ve RIPK3 hemen hemen aynı amiloid benzeri desenler (şerit 2 ve 4, şekil 1) gösterdi. Ancak, MLKL lifler daha heterojen ve RIPK1/RIPK3 lifleri (lane 6, şekil 1) küçük olmak gibiydi. Bizi ister MLKL RIPK1/RIPK3-bağımsız lifler oluşturur ya da MLKL sade bir şekilde büyük RIPK1/RIPK3/MLKL lifleri SDD-yaş sırasında ayrışıp. olasılığını adrese 2D SDD-yaş yöntem geliştirm...

Watch this Scientific Journal Video about Use of Two Dimensional Semi-denaturing Detergent Agarose Gel Electrophoresis to Confirm Size Heterogeneity of Amyloid or Amyloid-like Fibers at JoVE.com

Use of Two Dimensional Semi-denaturing Detergent Agarose Gel Electrophoresis to Confirm Size Heterogeneity of Amyloid or Amyloid-like Fibers

İki boyutlu yarı denaturing deterjan özel Jel Elektroforez amiloid veya amiloid benzeri liflerin boyutu heterojen onaylamak için kullanımı

Burada biz iki boyutlu yarı denaturing özel Jel Elektroforez amiloid benzeri lifler türdeş olmayan boyutta varlığını doğrulamak ve amiloid liflerinin ayrışma sırasında jel boyutu heterojenite kaynaklanmaktadır olasılığını dışlamak için kullanın koşma oluşum.

Amyloid or amyloid-like fibers have been associated with many human diseases, and are now being discovered to be important for many signaling pathways. ...

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Research

JoVE Journal

Biochemistry

9.7K Görüntüleme.  UT Southwestern Medical Center. Burada biz iki boyutlu yarı denaturing özel Jel Elektroforez amiloid benzeri lifler türdeş olmayan boyutta varlığını doğrulamak ve amiloid liflerinin ayrışma sırasında jel boyutu heterojenite kaynaklanmaktadır olasılığını dışlamak için kullanın koşma oluşum.

Video: Use of Two Dimensional Semi-denaturing Detergent Agarose Gel Electrophoresis to Confirm Size Heterogeneity of Amyloid or Amyloid-like Fibers

SDS-PAGE/Immunoblot Detection of A&beta; Multimers in Human Cortical Tissue Homogenates using Antigen-Epitope Retrieval

The anomalous folding and polymerization of the &beta;-amyloid (A&beta;) peptide is thought to initiate the neurodegenerative cascade in Alzheimer's disease pathogenesis1. A&beta; is predominantly a 40- or 42-amino acid peptide that is prone to self-aggregation into &beta;-sheet-rich amyloid fibrils that are found in the cores of cerebral senile plaques in Alzheimer's disease. Increasing evidence suggests that low molecular weight, soluble A&beta; multimers are more toxic than fibrillar A&beta; amyloid2. The identification and quantification of low- and high-molecular weight multimeric A&beta; species in brain tissue is an essential objective in Alzheimer's disease research, and the methods employed also can be applied to the identification and characterization of toxic multimers in other proteopathies3. Naturally occurring A&beta; multimers can be detected by SDS-polyacrylamide gel electrophoresis followed by immunoblotting with A&beta;-specific antibodies. However, the separation and detection of multimeric A&beta; requires the use of highly concentrated cortical homogenates and antigen retrieval in small pore-size nitrocellulose membranes. Here we describe a technique for the preparation of clarified human cortical homogenates, separation of proteins by SDS-PAGE, and antigen-epitope retrieval/Western blotting with antibody 6E10 to the N-terminal region of the A&beta; peptide. Using this protocol, we consistently detect A&beta; monomers, dimers, trimers, tetramers, and higher molecular weight multimers in cortical tissue from humans with Alzheimer's pathology.

SDS-PAGE/Immunoblot Detection of A&#946; Multimers in Human Cortical Tissue Homogenates using Antigen-Epitope Retrieval

We describe a technique for the preparation of clarified human cortical homogenates, protein separation by SDS-PAGE, antigen retrieval and immunoblotting with an antibody to the A&beta; peptide. Using this protocol, we consistently detect monomeric and multimeric A&beta; in cortical tissue from humans with Alzheimer's pathology.

İnsan Kortikal Doku Aβ multimerlerin SDS-PAGE/Immunoblot Algılama Antijen-epitop Alma kullanarak homojenatlarında

The anomalous folding and polymerization of the &beta;-amyloid (A&beta;) peptide is thought to initiate the neurodegenerative cascade in Alzheimer's ...

Nörobilim

Screening for Amyloid Aggregation by Semi-Denaturing Detergent-Agarose Gel Electrophoresis

Amyloid aggregation is associated with numerous protein misfolding pathologies and underlies the infectious properties of prions, which are conformationally self-templating proteins that are thought to have beneficial roles in lower organisms.  Amyloids have been notoriously difficult to study due to their insolubility and structural heterogeneity.  However, resolution of amyloid polymers based on size and detergent insolubility has been made possible by Semi-Denaturing Detergent-Agarose Gel Electrophoresis (SDD-AGE).  This technique is finding widespread use for the detection and characterization of amyloid conformational variants.  Here, we demonstrate an adaptation of this technique that facilitates its use in large-scale applications, such as screens for novel prions and other amyloidogenic proteins.  The new SDD-AGE method uses capillary transfer for greater reliability and ease of use, and allows any sized gel to be accomodated.  Thus, a large number of samples, prepared from cells or purified proteins, can be processed simultaneously for the presence of SDS-insoluble conformers of tagged proteins.

SDD-AGE is a useful technique for the detection and characterization of amyloid-like polymers in cells. Here we demonstrate an adaptation that makes this technique amenable to large-scale applications.

Yarı denatüre Deterjan-Agaroz Jel Elektroforez Amiloid Toplama taraması

Amyloid aggregation is associated with numerous protein misfolding pathologies and underlies the infectious properties of prions, which are ...

Biyoloji

Consensus Brain-derived Protein, Extraction Protocol for the Study of Human and Murine Brain Proteome Using Both 2D-DIGE and Mini 2DE Immunoblotting

Two-dimensional gel electrophoresis (2DE) is a powerful tool to uncover proteome modifications potentially related to different physiological or pathological conditions. Basically, this technique is based on the separation of proteins according to their isoelectric point in a first step, and secondly according to their molecular weights by SDS polyacrylamide gel electrophoresis (SDS-PAGE). In this report an optimized sample preparation protocol for little amount of human post-mortem and mouse brain tissue is described. This method enables to perform both two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and mini 2DE immunoblotting. The combination of these approaches allows one to not only find new proteins and/or protein modifications in their expression thanks to its compatibility with mass spectrometry detection, but also a new insight into markers validation. Thus, mini-2DE coupled to western blotting permits to identify and validate post-translational modifications, proteins catabolism and provides a qualitative comparison among different conditions and/or treatments. Herein, we provide a method to study components of protein aggregates found in AD and Lewy body dementia such as the amyloid-beta peptide and the alpha-synuclein. Our method can thus be adapted for the analysis of the proteome and insoluble proteins extract from human brain tissue and mice models too. In parallel, it may provide useful information for the study of molecular and cellular pathways involved in neurodegenerative diseases as well as potential novel biomarkers and therapeutic targets.

A common protein extraction protocol using urea/thiourea/SDS buffer for human and mice brain tissue allows indentification of proteins by 2D-DIGE and their subsequent characterization by mini 2DE immunoblotting. This method enables one to obtain more reproducible and reliable results from human biopsies and experimental models.

Konsensus Beyin kaynaklı protein, 2D-DIGE ve Mini 2DE İmmünoblotting Hem kullanma İnsan ve Kemirgen Beyin proteomun Çalışmaları Ekstraksiyon Protokolü

Two-dimensional gel electrophoresis (2DE) is a powerful tool to uncover proteome modifications potentially related to different physiological or ...

Biochemical Purification and Proteomic Characterization of Amyloid Fibril Cores from the Brain

Proteinaceous fibrillar inclusions are key pathological hallmarks of multiple neurodegenerative diseases. In the early stages of Alzheimer&#39;s disease (AD), amyloid-beta peptides form protofibrils in the extracellular space, which act as seeds that gradually grow and mature into large amyloid plaques. Despite this basic understanding, current knowledge of the amyloid fibril structure, composition, and deposition patterns in the brain is limited. One major barrier has been the inability to isolate highly purified amyloid fibrils from brain extracts. Affinity purification and laser capture microdissection-based approaches have been previously used to isolate amyloids but are limited by the small quantity of material that can be recovered. This novel, robust protocol describes the biochemical purification of amyloid plaque cores using sodium dodecyl sulfate (SDS) solubilization with sucrose density gradient ultracentrifugation and ultrasonication and yields highly pure fibrils from AD patients and AD model brain tissues. Mass spectrometry (MS)-based bottom-up proteomic analysis of the purified material represents a robust strategy to identify nearly all the primary protein components of amyloid fibrils. Previous proteomic studies of proteins in the amyloid coronae have revealed an unexpectedly large and functionally diverse collection of proteins. Notably, after refining the purification strategy, the number of co-purifying proteins was reduced by more than 10-fold, indicating the high purity of the isolated SDS insoluble material. Negative staining and immuno-gold electron microscopy allowed confirmation of the purity of these preparations. Further studies are required to understand the spatial and biological attributes that contribute to the deposition of these proteins into amyloid inclusions. Taken together, this analytical strategy is well-positioned to increase the understanding of amyloid biology.

This biochemical purification method with mass spectrometry-based proteomic analysis facilitates the robust characterization of amyloid fibril cores, which may accelerate the identification of targets for preventing Alzheimer's disease.

Beyinden Amiloid Fibril Çekirdeklerinin Biyokimyasal Saflaştırılması ve Proteomik Karakterizasyonu

Proteinaceous fibrillar inclusions are key pathological hallmarks of multiple neurodegenerative diseases. In the early stages of Alzheimer&#39;s disease ...

Separating Protein with SDS-PAGE

Sodium Dodecyl Sulfate Poly-Acrylamide Gel Electrophoresis, or SDS-PAGE, is a widely-used technique for separating mixtures of proteins based on their size and nothing else. SDS, an anionic detergent, is used to produce an even charge across the length of proteins that have been linearized. By first loading them into a gel made of polyacrylamide and then applying an electric field to the gel, SDS-coated proteins are then separated. The electric field acts as the driving force, drawing the SDS coated proteins towards the anode with larger proteins moving more slowly than small proteins. In order to identify proteins by size, protein standards of a known size are loaded along with samples and run under the same conditions.
This video presents an introduction to SDS-PAGE by first explaining the theory behind it and later demonstrating its step-by-step procedure. Various experimental parameters, such as the polyacrylamide concentration and voltage applied to the gel are discussed. Downstream staining methods like Coomassie and silver stains are introduced, and variations of the method, like 2D gel electrophoresis are presented.

Separating Protein with SDS-PAGE: Procedure and Applications

Sodium Dodecyl Sulfate Poly-Acrylamide Gel Electrophoresis, or SDS-PAGE, is a widely-used technique for separating mixtures of proteins based on their ...

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Hücresel ve Moleküler Biyolojide Temel Yöntemler

Two-Dimensional Gel Electrophoresis

Two-dimensional gel electrophoresis (2DGE) is a technique that can resolve thousands of biomolecules from a mixture. This technique involves two distinct separation methods that have been coupled together: isoelectric focusing (IEF) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). This physically separates compounds across two axes of a gel by their isoelectric points (an electrochemical property) and their molecular weights.
The procedure in this video covers the main concepts of 2DGE and a general procedure for characterizing the composition of a complex protein solution. Three examples of this technique are shown in the applications section, including biomarker detection for disease initiation and progress, monitoring treatment in patients, and the study of proteins following posttranslational modification (PTM).
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Two-dimensional, or 2D, gel electrophoresis is a technique utilizing two distinct separation methods which can separate thousands of proteins from a single mixture. One of the techniques, SDS-PAGE or sodium dodecyl sulfate polyacrylamide gel electrophoresis, cannot fully separate complex mixtures alone. 2D gel electrophoresis couples the SDS-PAGE to a second method, isoelectric focusing or IEF, which separates based on isoelectric points, allowing for the resolution of potentially all proteins in a cell lysate. This video will show the principles of 2D gel electrophoresis, a general procedure, and some of its biomedical applications.
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2D gel electrophoresis begins with IEF as the first dimension. Every protein has a pH value, called the isoelectric point or pI, where the net charge is zero. When a protein is subjected to an electric field, it will move toward the electrode with opposite charge. Samples of interest are loaded onto immobilized pH gradient, or IPG, strips which have embedded ampholytes, molecules containing both acidic and basic groups. An electric field is then applied to the pH gradient strip, causing the proteins to migrate until they reach the pH value matching their pI, where they lose their net charge.
Prior to running the second-dimension, the embedded proteins are treated with SDS, denaturing them and providing a uniform negative charge. Once completed, the IPG strips are placed onto a polyacrylamide gel. An applied electric field draws the proteins toward the anode, with larger proteins moving more slowly through the gel.
Once the protein mixture has been separated according to pI and molecular weight, the proteome map is visualized using stains, and proteins of interest are identified.
Now that we have discussed the principles of 2D gel electrophoresis, let&#39;s go over a typical laboratory procedure.
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Before the experiment can be performed, the proteins must be solubilized into media. Solubilization of the sample is achieved by de-aggregating proteins with a combination of chaotropic agents for disrupting hydrogen-bonding interactions, nonionic detergents to prevent altering of the proteins&#39; charge, reducing agents to break disulfide bonds, and buffers. To remove interfering abundant proteins and other molecules, the material is sequentially extracted by centrifugation, and collection of the resulting pellet; followed by treatment with endonuclease, an enzyme used to consume any DNA that would interfere with the experiment.
Once the proteins have been solubilized, IPG strips are prepared by rinsing with a strip-cleaning solution and left upside-down to dry. Each strip is then assigned a strip holder number. Once ready, the cellular extract is loaded onto the strips in a slow, sliding motion from the negative to the positive end. For the purpose of rehydration, damp blotting paper is placed on top of the electrode and under the gel strips; the IPG strips are then lined onto the IEF instrument. A high electric current is applied, and the proteins begin to migrate.
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Following completion of the first dimension, the gel for SDS-PAGE is prepared in a casting apparatus. The IPG strips are treated by placing them face down in SDS-containing equilibration buffer. The electrophoresis unit is readied with the addition of electrophoresis buffer. The treated IPG strips are collected using tweezers, placed on top of the gel plates, and sealed with agarose-sealing solution. A voltage source then applies an electric field, which is held until the fastest-moving proteins are 1 cm from the bottom of the gel.
After completion of the electrophoresis, the proteins must be visualized. Traditionally this is performed by staining with Coomassie blue or silver nitrate. Proteins of interest may be transferred from the gel, and analyzed by Western blot analysis.
A second identification approach involves excision of the proteins from the gel, digesting them, than analyzing them by mass spectrometry.
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Now that we&#39;ve reviewed a procedure, let&#39;s look at some of the uses for 2D gel electrophoresis.
One of the most common uses for this technique is the identification of molecules involved in disease initiation and progression. 2D gel electrophoresis, coupled with mass spectrometry, can detect the up- or down-regulation of specific proteins in diseased areas in comparison to healthy ones.
Additionally, 2D gel electrophoresis is useful in following the progress of patients&#39; response to a potential therapeutic drug. Specimens may be taken from patients at various timepoints following administration of the treatment. In this way, 2D gel electrophoresis coupled with Western blot or mass spectrometry analysis, can detect proteins associated with negative responses such as inflammation; or the absence of proteins in an alleviated state.
Another use for 2D gel electrophoresis is in the study of protein structure and function following posttranslational modification, or PTM, which are additions to proteins following their translation from mRNA. PTM&#39;s can regulate a variety of functions, including protein signaling, gene expression, or cause oxidative damage. 2D gel electrophoresis is sensitive to modifications such as methylation or acetylation, which can cause a shift in pI as well as the molecular weight.
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You&#39;ve just watched JoVE&#39;s video on 2D gel electrophoresis. This video described the principles of the technique, a typical experimental procedure, and several of its applications in the field of biomedicine.
Thanks for watching!
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Two-dimensional gel electrophoresis (2DGE) is a technique that can resolve thousands of biomolecules from a mixture. This technique involves two distinct ...

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Biyokimya

Isolation of Amyloid Fibrils from Brain Tissue Extract

Source: Upadhyay, A. et al., <a href="https://app.jove.com/v/63816">Biochemical Purification and Proteomic Characterization of Amyloid Fibril Cores from the Brain.</a> J. Vis. Exp. (2022)
This video demonstrates the protocol for isolating&#160; amyloid fibrils from mouse brain tissue extract using density gradient centrifugation and digestion of non-amyloid proteins.

Beyin Dokusu Ekstraktından Amiloid Fibrillerin İzolasyonu

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Tissue harvesting and ...

JoVE Encyclopedia of Experiments

Neuropathology

Neurodegenerative Diseases

Two-Dimensional Semi-Denaturing Agarose Gel Electrophoresis: A Technique to Separate Polymorphic Amyloids Fibers Based on Size Heterogeneity

Source: Hanna-Addams, S., et al. <a href="https://www.jove.com/v/57498">Use of Two Dimensional Semi-denaturing Detergent Agarose Gel Electrophoresis to Confirm Size Heterogeneity of Amyloid or Amyloid-like Fibers</a>. J. Vis. Exp. (2018).
This video demonstrates the two-dimensional semi-denaturing detergent agarose gel electrophoresis-based separation of amyloid fibers. The technique separates the polymorphic amyloid aggregates based on their heterogeneous size.

İki Boyutlu Yarı Denatüre Edici Agaroz Jel Elektroforezi: Polimorfik Amiloid Liflerini Boyut Heterojenliğine Göre Ayırmak İçin Bir Teknik

Source: Hanna-Addams, S., et al. Use of Two Dimensional Semi-denaturing Detergent Agarose Gel Electrophoresis to Confirm Size Heterogeneity of Amyloid or ...

Biyolojik Teknikler

Elektroforez Teknikleri

Two-Dimensional Polyacrylamide Gel Electrophoresis: A Separation Technique for Analysis of Proteins in Complex Protein Mixture Based on Charge and Molecular Weight

Source: Zhan, X., et al. <a href="https://www.jove.com/v/56739">Two-dimensional Gel Electrophoresis Coupled with Mass Spectrometry Methods for an Analysis of Human Pituitary Adenoma Tissue Proteome</a> J. Vis. Exp. (2018)
In this video, we demonstrate a two-dimensional gel electrophoresis method that separates individual proteins from a complex protein mixture obtained from tumor tissues of human nonfunctional pituitary adenoma (NFPA). The proteins are separated based on their electric charge in the first dimension and further separated based on molecular weights in the second dimension.

İki Boyutlu Poliakrilamid Jel Elektroforezi: Yük ve Moleküler Ağırlığa Dayalı Kompleks Protein Karışımındaki Proteinlerin Analizi İçin Bir Ayırma Tekniği

Source: Zhan, X., et al. Two-dimensional Gel Electrophoresis Coupled with Mass Spectrometry Methods for an Analysis of Human Pituitary Adenoma Tissue ...

Two-dimensional Gel Electrophoresis

Two-dimensional gel electrophoresis is a high-resolution protein separation method first introduced by O&#39;&#160;Farrell and Klose in 1975. This method involves protein separation by two dimensions, mass and charge, making it more accurate than one-dimensional gel electrophoresis.
The first dimension separation uses the isoelectric focusing or IEF technique performed on immobilized pH gradient (IPG) strips that separate proteins according to their isoelectric points.
Biological samples, such as&#160; cells and tissues, are first prepared for IEF by treatment with a buffer containing urea, dithiothreitol, detergents, and ampholytes. Here, urea and detergent solubilize and denature the proteins. Dithiothreitol is a reducing agent that cleaves disulfide bonds. Ampholytes used in a suitable concentration solubilize the protein and maintain pH. The buffer also rehydrates the IPG strip that helps it absorb the proteins before their charge-based separation.
Proteins are amphoteric molecules with positive and negative charges. The amphoteric protein molecules migrate on the pH gradient of the IPG strip under the electric current. Once the proteins reach their isoelectric point, they immobilize because, at this point, they have no net charge. IPG strips have a fixed pH gradient; for instance, strips with a smaller pH range (for example, pH 4-7) can detect proteins with higher accuracy than those with a broader range (pH 3-10). Once the proteins separate, the IPG strips are treated with sodium dodecyl sulfate (SDS) buffer to proceed to the next phase, i.e., mass-based separation using SDS-PAGE (polyacrylamide gel electrophoresis). SDS covers the uncharged protein on the IPG strips with a negative charge, and glycerol reduces electroendosmosis which helps the transfer of proteins from the first to the second dimension.
Generally, bands on the gel can be visualized upon staining. Sometimes radiolabelled samples are used to obtain an autoradiographic image. Samples capable of producing fluorescent images can also be used.
Two-dimensional gel electrophoresis has gained importance owing to the precision of the outcome results. However, the method is unsuitable for distinguishing highly hydrophobic proteins,&#160; proteins with high molecular mass, or those present in very low quantities per cell.

İki Boyutlu Jel Elektroforezi

Two-dimensional gel electrophoresis is a high-resolution protein separation method first introduced by O&#39;&#160;Farrell and Klose in 1975. This method ...