Bu eser NIH (RO1-NS090390) tarafından desteklenmiştir. Ayrıca Dr. Joan Steffanfor teknik yardım ve tartışma bu tahlil geliştirilmesi sırasında teşekkür etmek istiyorum.

Hayvan etik beyanı - deneyler yürütülen Kılavuzu ile sıkı uygun bakım ve kullanım laboratuvar hayvanları Ulusal Sağlık enstitüleri ve kurumsal hayvan bakım ve kullanım Komitesi () tarafından onaylanmış hayvan araştırma protokolü IACUC) University of California, Irvine, bir AAALAC akredite kurum. Tüm çabaları hayvan acı en aza indirmek için yapılmıştır.
1. lizis arabellekleri hazırlanması
<ol><li>"Çözünür" lizis arabellek hazırlamak (10 mM Tris pH 7.4, % 1 Triton-X 100, 150 mM NaCl, % 10 gliserol) ve 0,22 µm filtresi ile steril filtre.<ol>
<li>Çalışma "Çözünür" lizis için bir tampon, N-Ethylmaleimide (NEM) ölçmek ve iyice 100 mM phenylmethysulfonyl flor (PMSF) % 100 çözünmüş olarak dağıtılması alkol. Not: Lizis arabellek inhibitörleri: (i) 20 mM N-ethylmaleimide (NEM); (ii) 0.2 mM PMSF; (iii) 1 mM sodyum orthovanadate (Na3VO4); (iv) 1 µg/mL leupeptin; (v) 1 µg/mL aprotinin; (vi) 20 mM sodyum florür (kaf). Dikkat: bir yüz maskesi NEM boyunca protokolünün çalışırken giyiyorum. Lizis arabellek çalışma aktif inhibitörleri sağlamak için taze yapılmalıdır.</li>
		<li>Proteaz inhibitörleri ile çözünür lizis tampon için inhibitörleri aşağıdaki sırayla ekleyin: sodyum florür (kaf), soğuk lizis arabellek, sodyum orthovanadate (Na3VO4), Aprotinin ve Leupeptin bir birim kadar.</li>
	</ol>
</li>
	<li>"Çözünmez" lizis arabellek için % 4 SDS "Soluble" lizis arabellek için 2 mL % 20 SDS 500 µL ekleyerek ek. Oda sıcaklığında bırakın. Not: 4 ° C'de tutulur dışarı SDS çökelti; Bu oda sıcaklığında tutmak. Son lizis arabellek oluşturma: 10 mM Tris (pH 7,4); % 1 Triton X-100; 150 mM NaCl; % 10 gliserol; %4 SDS (sadece çözünmez).</li>
</ol>2. çözünür/çözünmez ayırma iletişim kuralı
Not: Bkz. şekil 1 bir şematik için.
<ol><li>Tüpler her örnek için iki farklı etiket: "Soluble" ve "Çözünmez."</li>
	<li>X-µL buz gibi çalışma çözünür lizis arabelleği proteaz inhibitörleri (bkz: 1.1.1) doku ile takıma ekleyin. Not: X = birim değişir doku miktarı bağlı olarak; Bkz. Tablo 1.</li>
	<li>(Yani, striatum) fare beyin doku için dounce cam 1 mL douncer ve damlalıklı "Çözünmez" içine yavaş yavaş 30 kere tüp (buz üzerinde tutun) etiketli. Not: bubbles formu olacak ve eksik homojenizasyon verim gibi douncing, başlattıktan sonra sıvı yüzeyinin kırmamaya dikkat et.</li>
	<li>(Yani, HeLa, HEK293T) hücreler için hücreleri bir kez soğuk, 1 x PBS ile yıkayın. PBS kaldırmak ve asgari miktar lizis arabelleği hücrelere uygulayın ve hücre sıyırıcı kaldırın. Not: İçin yukarıdaki hücre satır, hücre 6 iyi plaka başına 5 x 105 hücre, kaplama ve confluency yakın büyüdü. Bir tane de fare striatum için kullanılan birimleri karşılaştırılabilir.</li>
	<li>Homogenate veya hücre içine lysate etiketli, "Çözünmez" tüpler aktarmak ve buz 1 h (son tüp işlendikten sonra başlangıç saat) koşullar. Not: cep lysates için kuluçka başlamadan önce hücre kümeleri bölmek için birkaç kez triturate. Kabarcıklar oluşumunu önlemek. Kısaca girdap darbe her örnek için 1 s lysing aracılığıyla yarı yolda.</li>
	<li>15.000 x g 20 dk için de 4 ° C'de örnekler santrifüj kapasitesi.</li>
	<li>Süpernatant "Soluble" kesir olarak kurtarmak.<ol>
<li>Damlalıklı ile kaldırmak için bu kesir kontamine gibi Pelet/bulutlu katman bozmamaya dikkat. Damlalıklı çözünür kesir "Soluble" içine tüp etiketli. Buz üstünde tutun.</li>
	</ol>
</li>
	<li>Her yıkadıktan sonra Pelet 500 µL lizis arabellek (x2) ve santrifüj ile 15.000 x g 5 min için de 4 ° C'de yıkama; Bulutlu katman bazıları kapalı pipetted sorun değil.</li>
	<li>Son yıkama sonra kalan tüm yıkama arabellek yalnızca doku Pelet bırakarak kaldırın. Not: Bu noktadan sonra çözünmez örnekleri şimdi oda sıcaklığında tutulmalıdır.</li>
	<li>Resuspend Pelet X-µL lizis arabellek % 4 SDS ile takviye ile (birim Pelet boyutuna bağlı olarak ayarlanabilir; bkz. Tablo 1).</li>
	<li>Her örnek için 30 solüsyon içeren temizleyicide s bir sonda sonicator ile oda sıcaklığında (125 Watt, 20 kHz, nabız, genlik % 40, bkz: Malzemeler tablo).</li>
	<li>(Haddeleme kaynatın) örnekleri için 30 dk kaynatın; Kısaca santrifüj kapasitesi (15 s) 6.000 x g herhangi bir örnek kaybetmemek için de.</li>
	<li>DC (deterjan uyumlu) protein tahlil gerçekleştirmek ( Tablo malzemelerigörmek) veya Lowry protein tahlil protein konsantrasyonu (1/5 ilgili lizis arabelleği hücre kültürü için örnek ve 1/10 için doku lysates başlamış dilutions önerilen) ölçmek için. Not: Protein konsantrasyonları yüksek deterjan konsantrasyon nedeniyle DC veya Lowry tahlil kullanarak okunması gerekir.</li>
	<li>Donma-çözülme sorunları önlemek için 50 µL toplu işleme aliquot örnekleri. Not: İletişim kuralı burada daha fazla biyokimyasal analiz önce her iki kesirler-20 ° C'de depolayarak duraklatılmış</li>
</ol>3. Western Blot ve miktar
<ol><li>Batı açıkları olarak bildirilen başka bir yerde1,13 30 µg sulandrarak Örnek 1:1 arabellek (boya, 1 azaltmak x yükleme x 1.6) yük içinde yerine getirilir. Not: protein fraksiyonu başına 30 µg yeterlidir, ancak en iyi çözünürlük için ayarlanabilir. 3 - %8 Tris-asetat Poli-Akrilamid jelleri çözünmez kesir için kullanılmalıdır. 4 - %12 bis-Tris jelleri çözünür kesir için tavsiye edilir.</li>
	<li>Kısaca 5 dk. santrifüj örnekleri (haddeleme kaynatın) kaynatın (15 s) örnek toplamak için 6.000 x g de.</li>
	<li>Kesirler SDS-sayfa ve western blot, azaltılmış örnekleri için üreticinin protokolüne göre analiz. Not: Çözünmez kesir en iyi 0,45 µM nitroselüloz membran Transfer edildiğinde giderir. 0,45 veya 0.2 µM nitroselüloz faiz protein büyüklüğüne bağlı olarak çözünür kesir için kullanılabilir. Bazı optimizasyon HMW malzemeleri en iyi transferi için gerekli olabilir.</li>
	<li>Membran protein verimlilik yükleme görselleştirmek için tüm protein leke ile (örneğin, MEMcode, bkz: Malzemeler tablo) leke. Not: Batı lekesi ortalama piksel yoğunluğu tarafından çözümlenebilir. Çözünür kesirler en iyi deneysel sistemi için uygun bir ev tutma protein için normalleştirilmiş. Çözünmez kesir olarak göreli protein bereket tersinir protein boyama veya ayrı leke üzerinde Histon 3 normalleştirme tarafından doğrulandı.</li>
</ol>

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Yazarlar ifşa gerek yok.

Bazı önlemler tutarlı ve kantitatif sonuç sağlamak yukarıdaki iletişim kuralları için ihtiyaç vardır. İlk olarak, mHTT her iki kesirler kendiliğinden toplamları üzerine birden çok donma çözülme çevrimleri, zamanla oluşturacak özellikle ne zaman yüksek bir konsantrasyon içinde. Böylece protein preps aliquots dondurma ve yalnızca gerekli birim Protokolü yukarıda açıklandığı gibi tahlil çalıştırmadan önce çözülme çok önemlidir. Ayrıca, çözünmez kesir çözdürme üzerine beyaz ...

Uygun katlama sağlamak protein kalite kontrol ağları bozulma ve yıkımı proteinlerin patoloji Alzheimer hastalığı (Ah), Parkinson hastalığı (PD), Huntington hastalığı (HD) için büyük olasılıkla orta ve protein diğer "misfolding" olduğunu 2,3bozuklukları. Proteostasis ağ bileşenleri ve onların katkıları patoloji için detaylı bir anlayış bu nedenle geliştirilmiş tedavi müdahaleler geliştirmek için çok önemli. HD bir CAG tekrar polyglutamines (polyQ) huntingtin (HTT) protein4genişletilmiş bir streç sonuçlanan HD gen içinde anormal genişlemesi nedeniyle oluşur. Bir tutarlı patolojik HD Patogenez elde edilen misfolding, birikimi ve çeşitli şekillerde normal hücre homeostazı ve işlev girişimine gösterilen HTT beyin bölgelerinde ve periferik dokularda agregasyon özelliğidir 5 , 6. iken HTT ubiquitously ifade, striatum orta dikenli nöronların Ayrıca HD patogenezi ile ilgili dikkate değer kortikal Atrofi ile seçmeli olarak savunmasız ve en açıktan etkilenen vardır.
HTT HD hasta ve hayvan modelleri beyinde agrega oluşumu genellikle hastalık ilerleme ve baskın negatif kazanç mHTT7için işlevinin bir proxy marker olarak hizmet vermiştir. Hangi protein tarafından misfolding ve toplama mHTT kaynaklı toksisite için katkıda bulunabilir kesin mekanizmaları belirsiz kalır rağmen beyindeki bu kapanımlar HD hasta ve çeşitli hayvan modelleri oluşumu bir sabit ve kaçınılmaz özelliğidir. N-terminal içeren büyük ölçüde birikmiş HTT parçaları oluşan için o proteolizis belirten polyQ, genişletilmiş ve tam uzunlukta HTT yanı sıra alternatif Uçbirleştirme8,işleme9 oynayabilir kapanımlar görünür bir HD. N-terminal HTT parçaları patogenezinde önemli rol hızla toplamak, nucleate ve hızlandırmak veya toplama işlemi10yaymak HTT patolojik bir tür teşkil edebilir.
Ancak, bu eklemeler varlığı mutlaka HTT kaynaklı toksisite ya da hücre ölüm11ile ilişkilendirmek değil. HTT çözünür bir monomer çözünür oligomeric türler ve β-yapraklık liflerinde bir toplama işleminden çözünmez toplamları ve kapanımlar12geçmesi teklif edildi. Bu protein türleri standart biyokimyasal deneyleri ile çözme onların istikrar içinde düşük-deterjan lizis tampon ve standart biyokimyasal testleri kullanarak görselleştirme zorluk nedeniyle alandaki bir mücadele vardı. Böylece, metodolojik konusu derecesi ve birikmiş ve toplanan mHTT desen saptamada kritik önem taşır.
Burada sunulan Protokolü HTT, özellikle HD Patogenez ve hastalık ilerleme1,13 ile yakından izlemek için görünür bir çözünmez HMW HTT türlerin oluşumu, birkaç ara ürün görselleştirmek için bir yöntem sağlar ,14. Gidermek ve mHTT birden çok tür takip edememek araştırmacılar hastalığın patogenezinde çalışma ve modülasyon ve hastalık patogenezinde üzerindeki etkisi ile potansiyel terapötik müdahaleler değerlendirmek biyokimyasal bir araçtır.

<table>
	<tbody>
		<tr>
			<td>Sterile Filter</td>
			<td>Millipore</td>
			<td>SCGP505RE</td>
			<td>Screw cap, sterile vaccum filter</td>
		</tr>
		<tr>
			<td>1 mL Tissue Grinder, Dounce</td>
			<td>Wheaton</td>
			<td>357538</td>
			<td></td>
		</tr>
		<tr>
			<td>Sonicator</td>
			<td>Qsonica</td>
			<td>Model Q125</td>
			<td></td>
		</tr>
		<tr>
			<td>DC Protein Assay</td>
			<td>Biorad</td>
			<td>5000111</td>
			<td>Comparable to Lowry assay</td>
		</tr>
		<tr>
			<td>Tris 1M buffer solution</td>
			<td>Alfa Aesar</td>
			<td>J60636</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Fisher</td>
			<td>BP151-100</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl 5M solution</td>
			<td>Teknova</td>
			<td>S0251</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Fisher</td>
			<td>BP229-1</td>
			<td></td>
		</tr>
		<tr>
			<td>20% SDS solution</td>
			<td>Teknova</td>
			<td>S0295</td>
			<td></td>
		</tr>
		<tr>
			<td>N-ethylmaleimide</td>
			<td>Sigma</td>
			<td>E1271</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenylmethylsulfony flouride</td>
			<td>Sigma</td>
			<td>P7626</td>
			<td>create 100mM stock solution in 100%EtOH, store at 4&#176;C</td>
		</tr>
		<tr>
			<td>Sodium orthovanadate</td>
			<td>Sigma</td>
			<td>S6508</td>
			<td>Create 0.5M stock solution in water</td>
		</tr>
		<tr>
			<td>Leupeptin</td>
			<td>Sigma</td>
			<td>L2884</td>
			<td>Create 10mg/ml stock solution in water</td>
		</tr>
		<tr>
			<td>Aprotinin</td>
			<td>Sigma</td>
			<td>A1153</td>
			<td>Create 10mg/ml stock solution in water</td>
		</tr>
		<tr>
			<td>Sodium Fluoride</td>
			<td>Sigma</td>
			<td>S4504</td>
			<td>Create 500mM stock solution in water</td>
		</tr>
		<tr>
			<td>Anti-HTT</td>
			<td>Millipore</td>
			<td>MAB5492</td>
			<td>Use 1:1000 for western blot, 1:500 for filter retardation assay</td>
		</tr>
		<tr>
			<td>Anti-GAPDH</td>
			<td>Novus Biologicals</td>
			<td>NB100-56875</td>
			<td>Use 1:1000 for soluble western blot</td>
		</tr>
	</tbody>
</table>

fractionation for resolution of soluble and insoluble huntingtin species

Misfolded proteinleri birikimi patoloji Huntington hastalığı (HD) ve diğer birçok nörodejeneratif hastalıklar için merkezi bir noktada bulunuyor. Özellikle, bir ana patolojik HD yüksek molekül ağırlıklı kompleksleri içine mutant HTT (mHTT) protein birikimine anormal özelliğidir ve hücre içi dahil ceset parçaları ve diğer proteinler oluşur. Ölçmek ve mHTT içeren toplamları çeşitli formları katkılarıyla Floresans mikroskobu, western blot analizi ve filtre dahil anlamak için geleneksel yöntemler deneyleri tuzak.
Ancak, bu yöntemlerin çoğu belirli biçimi vardır ve bu nedenle mHTT protein akı toplama çözünürlük ve çözünürlük karmaşık yapısı nedeniyle tam durumunu çözmeyebilir.
Toplanan mHTT ve çeşitli değiştirilmiş form ve kompleksleri kimlik için ayırma ve solubilization hücresel toplamları ve parçaları zorunlu değildir. Burada biz yalıtmak ve görselleştirmek çözünür mHTT, monomerleri, reaksiyonlar, parçaları için bir yöntem tanımlamak ve çözünmez yüksek molekül ağırlıklı (HMW) mHTT türler birikmiş. HMW mHTT izler hastalık ilerlemesi ile fare davranışı çıktıları ile karşılık gelir ve beneficially belirli tedavi müdahaleler1tarafından modüle. Bu yaklaşım fare beyni, periferik doku ve hücre kültürü ile kullanılabilir ancak diğer sistemleri modellemek bağlamı olarak da bilinen hastalık için adapte edilebilir.

Kararlılık-in ayırma takip çözünür ve çözünmez hücre lysates Batı analizi ve filtre retardasyon deneyleri (Şekil 2) kullanarak tespit edilebilir. Örnek olarak, HEK293T hücre transfection reaktifi (örneğin, lipofectamine 2000) kullanarak transfected, ile HTT exon 1 kodlama cDNA 97 glutamin içeren Poli prolin zengin bölgeye göre takip15 tekrarlar ve bu hücreler için izin verildi 44 h. hücreleri tedavi edil...

Watch this Scientific Journal Video about Fractionation for Resolution of Soluble and Insoluble Huntingtin Species at JoVE.com

Fractionation for Resolution of Soluble and Insoluble Huntingtin Species

Fare beyin ve hücre kültür çözünür ve çözünmez mutant huntingtin türlerinin ayırma için bir yöntemi açıklanmıştır. Açıklanan yöntemi karakterizasyonu ve miktar huntingtin protein akı ve protein homeostazı hastalık patogenezinde ve tedirginlikler huzurunda analiz AIDS için yararlıdır

Çözünür ve çözünmez Huntingtin türler çözümlenmesi için ayırma

The accumulation of misfolded proteins is central to pathology in Huntington's disease (HD) and many other neurodegenerative disorders. Specifically, a ...

fractionation-for-resolution-soluble-insoluble-huntingtin

Research

JoVE Journal

Biochemistry

9.4K Görüntüleme.  University of California Irvine. Fare beyin ve hücre kültür çözünür ve çözünmez mutant huntingtin türlerinin ayırma için bir yöntemi açıklanmıştır. Açıklanan yöntemi karakterizasyonu ve miktar huntingtin protein akı ve protein homeostazı hastalık patogenezinde ve tedirginlikler huzurunda analiz AIDS için yararlıdır

Video: Fractionation for Resolution of Soluble and Insoluble Huntingtin Species

High-throughput Screening of Carbohydrate-degrading Enzymes Using Novel Insoluble Chromogenic Substrate Assay Kits

Carbohydrates active enzymes (CAZymes) have multiple roles in vivo and are widely used for industrial processing in the biofuel, textile, detergent, paper and food industries. A deeper understanding of CAZymes is important from both fundamental biology and industrial standpoints. Vast numbers of CAZymes exist in nature (especially in microorganisms) and hundreds of thousands have been cataloged and described in the carbohydrate active enzyme database (CAZy). However, the rate of discovery of putative enzymes has outstripped our ability to biochemically characterize their activities. One reason for this is that advances in genome and transcriptome sequencing, together with associated bioinformatics tools allow for rapid identification of candidate CAZymes, but technology for determining an enzyme's biochemical characteristics has advanced more slowly. To address this technology gap, a novel high-throughput assay kit based on insoluble chromogenic substrates is described here. Two distinct substrate types were produced: Chromogenic Polymer Hydrogel (CPH) substrates (made from purified polysaccharides and proteins) and Insoluble Chromogenic Biomass (ICB) substrates (made from complex biomass materials). Both CPH and ICB substrates are provided in a 96-well high-throughput assay system. The CPH substrates can be made in four different colors, enabling them to be mixed together and thus increasing assay throughput. The protocol describes a 96-well plate assay and illustrates how this assay can be used for screening the activities of enzymes, enzyme cocktails, and broths.

A high-throughput assay for enzyme screening is described. This multiplexed ready-to-use assay kit comprises of pre-chosen Chromogenic Polymer Hydrogel (CPH) substrates and complex Insoluble Chromogenic Biomass (ICB) substrates. Target enzymes are polysaccharide degrading endo-enzymes and proteases.

Yeni çözünmez Kromojenik altyapı deneyiyle kitleri kullanılarak karbonhidrat parçalayıcı Enzimlerin yüksek verimli tarama

Carbohydrates active enzymes (CAZymes) have multiple roles in vivo and are widely used for industrial processing in the biofuel, textile, detergent, paper ...

Biyokimya

Brain Membrane Fractionation: An Ex Vivo Approach to Assess Subsynaptic Protein Localization

Assessing the synaptic protein composition and function constitutes an important challenge in neuroscience. However, it is not easy to evaluate neurotransmission that occurs within synapses because it is highly regulated by dynamic protein-protein interactions and phosphorylation events. Accordingly, when any method is used to study synaptic transmission, a major goal is to preserve these transient physiological modifications. Here, we present a brain membrane fractionation protocol that represents a robust procedure to isolate proteins belonging to different synaptic compartments. In other words, the protocol describes a biochemical methodology to carry out protein enrichment from presynaptic, postsynaptic, and extrasynaptic compartments. First, synaptosomes, or synaptic terminals, are obtained from neurons that contain all synaptic compartments by means of a discontinuous sucrose gradient. Of note, the quality of this initial synaptic membrane preparation is critical. Subsequently, the isolation of the different subsynaptic compartments is achieved with light solubilization using mild detergents at differential pH conditions. This allows for separation by gradient and isopycnic centrifugations. Finally, protein enrichment at the different subsynaptic compartments (i.e., pre-, post- and extrasynaptic membrane fractions) is validated by means of immunoblot analysis using well-characterized synaptic protein markers (i.e., SNAP-25, PSD-95, and synaptophysin, respectively), thus enabling a direct assessment of the synaptic distribution of any particular neuronal protein.

Brain Membrane Fractionation: An Ex Vivo Approach to Assess Subsynaptic Protein Localization

Here, we present a brain membrane fractionation protocol that represents a robust procedure to isolate proteins belonging to different synaptic compartments.

Beyin Membranı Fraksiyonu: Bir<emEx Vivo</em&gt; Subsınaptik Protein Lokalizasyonunu Değerlendirmede Yaklaşım

Assessing the synaptic protein composition and function constitutes an important challenge in neuroscience. However, it is not easy to evaluate ...

Enrichment of Detergent-insoluble Protein Aggregates from Human Postmortem Brain

In this study, we describe an abbreviated single-step fractionation protocol for the enrichment of detergent-insoluble protein aggregates from human postmortem brain. The ionic detergent N-lauryl-sarcosine (sarkosyl) effectively solubilizes natively folded proteins in brain tissue allowing the enrichment of detergent-insoluble protein aggregates from a wide range of neurodegenerative proteinopathies, such as Alzheimer&#39;s disease (AD), Parkinson&#39;s disease and amyotrophic lateral sclerosis, and prion diseases. Human control and AD postmortem brain tissues were homogenized and sedimented by ultracentrifugation in the presence of sarkosyl to enrich detergent-insoluble protein aggregates including pathologic phosphorylated tau, the core component of neurofibrillary tangles in AD. Western blotting demonstrated the differential solubility of aggregated phosphorylated-tau and the detergent-soluble protein, Early Endosome Antigen 1 (EEA1) in control and AD brain. Proteomic analysis also revealed enrichment of &#946;-amyloid (A&#946;), tau, snRNP70 (U1-70K), and apolipoprotein E (APOE) in the sarkosyl-insoluble fractions of AD brain compared to those of control, consistent with previous tissue fractionation strategies. Thus, this simple enrichment protocol is ideal for a wide range of experimental applications ranging from Western blotting and functional protein co-aggregation assays to mass spectrometry-based proteomics.

An abbreviated fractionation protocol for the enrichment of detergent-insoluble protein aggregates from human postmortem brain is described.

Deterjan çözünmez Protein toplamları--dan insan öldükten sonra beyin zenginleştirme

In this study, we describe an abbreviated single-step fractionation protocol for the enrichment of detergent-insoluble protein aggregates from human ...

Quantitative and Qualitative Method for Sphingomyelin by LC-MS Using Two Stable Isotopically Labeled Sphingomyelin Species

We present a method of analyzing sphingomyelin (SM) qualitatively and quantitatively by liquid chromatography-electrospray Ionization-tandem mass spectrometry (LC-ESI-MS/MS). SM is a common sphingolipid composed of a phosphorylcholine and a ceramide as the hydrophilic and hydrophobic component, respectively. A number of SM species are present in mammalian cells due to a variety in the sphingoid long chain base (LCB) and an N-acyl moiety in the ceramide. In this report, we show a method of estimating the number of carbon and double bonds in a LCB and an N-acyl moiety based on their corresponding product ions in MS/MS/MS (MS3) experiments. In addition, we present a quantitative analysis method for SM using two stable isotopically labeled SM species, which facilitates determining the range used in SM quantitation. The present method will be useful in characterizing a variety of SM species in biological samples and industrial products such as cosmetics.

Here, we present a protocol to quantify and qualify each sphingomyelin species using multiple reaction monitoring and MS/MS/MS mode, respectively.

Sphingomyelin türler Sphingomyelin LC-MS iki kararlı Isotopically kullanarak tarafından için nicel ve nitel yöntemi etiketli

We present a method of analyzing sphingomyelin (SM) qualitatively and quantitatively by liquid chromatography-electrospray Ionization-tandem mass ...

Generation of Native, Untagged Huntingtin Exon1 Monomer and Fibrils Using a SUMO Fusion Strategy

Huntington&#39;s Disease (HD) is an inherited fatal neurodegenerative disease caused by a CAG expansion (&#8805;36) in the first exon of the HD gene, resulting in the expression of the Huntingtin protein (Htt) or N-terminal fragments thereof with an expanded polyglutamine (polyQ) stretch.
The exon1 of the Huntingtin protein (Httex1) is the smallest Htt fragment that recapitulates many of the features of HD in cellular and animal models and is one of the most widely studied fragments of Htt. The small size of Httex1 makes it experimentally more amenable to biophysical characterization using standard and high-resolution techniques in comparison to longer fragments or full-length Htt. However, the high aggregation propensity of mutant Httex1 (mHttex1) with increased polyQ content (&#8805;42) has made it difficult to develop efficient expression and purification systems to produce these proteins in sufficient quantities and make them accessible to scientists from different disciplines without the use of fusion proteins or other strategies that alter the native sequence of the protein. We present here a robust and optimized method for the production of milligram quantities of native, tag-free Httex1 based on the transient fusion of small ubiquitin related modifier (SUMO). The simplicity and efficiency of the strategy will eliminate the need to use non-native sequences of Httex1, thus making this protein more accessible to researchers and improving the reproducibility of experiments across different laboratories. We believe that these advances will also facilitate future studies aimed at elucidating the structure-function relationship of Htt as well as developing novel diagnostic tools and therapies to treat or slow the progression of HD.

Here, we present a robust and optimized protocol for the production of milligram quantities of native, tag-free monomers and fibrils of the exon1 of the Huntingtin protein (Httex1) based on the transient fusion of small ubiquitin related modifier (SUMO).

Yerli, etiketsiz Huntingtin Exon1 Monomer ve SUMO Fusion strateji kullanarak liflerinde nesil

Huntington&#39;s Disease (HD) is an inherited fatal neurodegenerative disease caused by a CAG expansion (&#8805;36) in the first exon of the HD gene, ...

Detection of Small GTPase Prenylation and GTP Binding Using Membrane Fractionation and  GTPase-linked Immunosorbent Assay

The Rho GTPase family belongs to the Ras superfamily and includes approximately 20 members in humans. Rho GTPases are important in the regulation of diverse cellular functions, including cytoskeletal dynamics, cell motility, cell polarity, axonal guidance, vesicular trafficking, and cell cycle control. Changes in Rho GTPase signaling play an essential regulatory role in many pathological conditions, such as&#160;cancer, central nervous system diseases, and immune system-dependent diseases. The posttranslational modification of Rho GTPases (i.e., prenylation by mevalonate pathway intermediates) and GTP binding are key factors which affect the activation of this protein. In this paper, two essential and simple methods are provided to detect a broad range of Rho GTPase prenylation and GTP binding activities. Details of the technical procedures that have been used are explained step by step in this manuscript.

Here we describe a protocol to investigate the prenylation and guanosine-5'-triphosphate (GTP)-loading of Rho GTPase. This protocol consists of two detailed methods, namely membrane fractionation and a GTPase-linked immunosorbent assay. The protocol can be used for measuring the prenylation and GTP loading of different other small GTPases.

Küçük GTPazlar prenilasyon ve bağlayıcı kullanarak membran ayırma ve GTPazlar bağlı Immunosorbent Assay GTP tespiti

The Rho GTPase family belongs to the Ras superfamily and includes approximately 20 members in humans. Rho GTPases are important in the regulation of ...

The Identification of Sea Lamprey Pheromones Using Bioassay-Guided Fractionation

Bioassay-guided fractionation is an iterative approach that uses the results of physiological and behavioral bioassays to guide the isolation and identification of an active pheromone compound. This method has resulted in the successful characterization of the chemical signals that function as pheromones in a wide range of animal species. Sea lampreys rely on olfaction to detect pheromones that mediate behavioral or physiological responses. We use this knowledge of fish biology to posit functions of putative pheromones and to guide the isolation and identification of active pheromone components. Chromatography is used to extract, concentrate, and separate compounds from the conditioned water. Electro-olfactogram (EOG) recordings are conducted to determine which fractions elicit olfactory responses. Two-choice maze behavioral assays are then used to determine if any of the odorous fractions are also behaviorally active and induce a preference. Spectrometric and spectroscopic methods provide the molecular weight and structural information to assist with the structure elucidation. The bioactivity of the pure compounds is confirmed with EOG and behavioral assays. The behavioral responses observed in the maze should ultimately be validated in a field setting to confirm their function in a natural stream setting. These bioassays play a dual role to 1) guide the fractionation process and 2) confirm and further define the bioactivity of isolated components. Here, we report the representative results of a sea lamprey pheromone identification that exemplify the utility of the bioassay-guided fractionation approach. The identification of sea lamprey pheromones is particularly important because a modulation of its pheromone communication system is among the options considered to control the invasive sea lamprey in the Laurentian Great Lakes. This method can be readily adapted to characterize the chemical communication in a broad array of taxa and shed light on waterborne chemical ecology.

Here, we present a protocol to isolate and characterize the structure, olfactory potency, and behavioral response of putative pheromone compounds of sea lampreys.

Bioassay-güdümlü ayırma kullanarak deniz taşemen Feromonlar tanımlaması

Bioassay-guided fractionation is an iterative approach that uses the results of physiological and behavioral bioassays to guide the isolation and ...

Cell Fractionation of U937 Cells in the Absence of High-speed Centrifugation

In this protocol we detail a method to obtain subcellular fractions of U937 cells without the use of ultracentrifugation or indiscriminate detergents. This method utilizes hypotonic buffers, digitonin, mechanical lysis and differential centrifugation to isolate the cytoplasm, mitochondria and plasma membrane. The process can be scaled to accommodate the needs of researchers, is inexpensive and straightforward. This method will allow researchers to determine protein localization in cells without specialized centrifuges and without the use of commercial kits, both of which can be prohibitively expensive. We have successfully used this method to separate cytosolic, plasma membrane and mitochondrial proteins in the human monocyte cell line U937.

Here, we present a protocol to isolate the plasma membrane, cytoplasm and mitochondria of U937 cells without the use of high-speed centrifugation. This technique can be used to purify subcellular fractions for subsequent examination of protein localization via immunoblotting.

Yüksek hızlı Santrifüjü yokluğunda U937 hücrelerinin hücre ayırma

In this protocol we detail a method to obtain subcellular fractions of U937 cells without the use of ultracentrifugation or indiscriminate detergents. ...

Measuring Interactions between Fluorescent Probes and Lignin in Plant Sections by sFLIM Based on Native Autofluorescence

In lignocellulosic biomass (LB), the activity of enzymes is limited by the appearance of non-specific interactions with lignin during the hydrolysis process, which maintains enzymes far from their substrate. Characterization of these complex interactions is thus a challenge in complex substrates such as LB. The method here measures molecular interactions between fluorophore-tagged molecules and native autofluorescent lignin, to be revealed by F&#246;rster resonance energy transfer (FRET). Contrary to FRET measurements in living cells using two exogenous fluorophores, FRET measurements in plants using lignin is not trivial due to its complex autofluorescence. We have developed an original acquisition and analysis pipeline with correlated observation of two complementary properties of fluorescence: fluorescence emission and lifetime. sFLIM (spectral and fluorescent lifetime imaging microscopy) provides the quantification of these interactions with high sensitivity, revealing different interaction levels between biomolecules and lignin.

This protocol describes an original setup combining spectral and fluorescence lifetime measurements to evaluate F&#246;rster resonance energy transfer (FRET) between rhodamine-based fluorescent probes and lignin polymer directly in thick plant sections.

Floresan Problar ve Lignin Bitki Kesitlerinde Doğal Otofloresan Dayalı SFLIM Ile Etkileşimlerinin Ölçülmesi

In lignocellulosic biomass (LB), the activity of enzymes is limited by the appearance of non-specific interactions with lignin during the hydrolysis ...

Conventional BODIPY Conjugates for Live-Cell Super-Resolution Microscopy and Single-Molecule Tracking

Single molecule localization microscopy (SMLM) techniques overcome the optical diffraction limit of conventional fluorescence microscopy and can resolve intracellular structures and the dynamics of biomolecules with ~20 nm precision. A prerequisite for SMLM are fluorophores that transition from a dark to a fluorescent state in order to avoid spatio-temporal overlap of their point spread functions in each of the thousands of data acquisition frames. BODIPYs are well-established dyes with numerous conjugates used in conventional microscopy. The transient formation of red-shifted BODIPY ground-state dimers (DII) results in bright single molecule emission enabling single molecule localization microscopy (SMLM). Here we present a simple but versatile protocol for SMLM with conventional BODIPY conjugates in living yeast and mammalian cells. This procedure can be used to acquire super-resolution images and to track single BODIPY-DII states to extract spatio-temporal information of BODIPY conjugates. We apply this procedure to resolve lipid droplets (LDs), fatty acids, and lysosomes in living yeast and mammalian cells at the nanoscopic length scale. Furthermore, we demonstrate the multi-color imaging capability with BODIPY dyes when used in conjunction with other fluorescent&#160;probes. Our representative results show the differential spatial distribution and mobility of BODIPY-fatty acids and neutral lipids in yeast under fed and fasted conditions. This optimized protocol for SMLM can be used with hundreds of commercially available BODIPY conjugates and is a useful resource to study biological processes at the nanoscale far beyond the applications of this work.&#160;

Conventional BODIPY conjugates can be used for live-cell single-molecule localization microscopy (SMLM) through exploitation of their transiently forming, red-shifted ground state dimers. We present an optimized SMLM protocol to track and resolve subcellular neutral lipids and fatty acids in living mammalian and yeast cells at the nanoscopic length scale.

Canlı Hücresüper çözünürlüklü mikroskopi ve Tek Moleküllü İzleme için Konvansiyonel BODIPY Konjugeleri

Single molecule localization microscopy (SMLM) techniques overcome the optical diffraction limit of conventional fluorescence microscopy and can resolve ...