Yazarlar maliyet eylem CM1406 kabul (epigenetik Kimya Biyoloji). Bu yayında bildirilen araştırma Pierre Mercier Vakfı tarafından desteklenmiştir.

1. hücre kültürü
Not: Standart doku kültürü mahallede aşağıdaki adımlar gerçekleştirilir. Steril tekniği ile aşinalık bekleniyor.
<ol>
	<li>HeLa hücreleri için %80 confluency % 10 fetal sığır serum, penisilin G takıma MEM bir T75 hücre kültür şişesi içinde kültür (100 U/mL) ve streptomisin (100 mg/mL). Not: Hücre kültürü, tedavi ve lizis adımları %5 CO2 steril koşullarda oksijen bir ortamda gerçekleştirilen 37 ° C'de hücre kuluçka adımlarla laminar akış altında yapılmaktadır.</li>
	<li>5 mL DPBS de iki kez hücrelerle yıkama, DPBS Aspire edin ve hücre ayrılma reaktif 1 mL ekleyin. Hücreleri ayırmak 5 min için 37 ° C'de kuluçkaya.</li>
	<li>MEM kültür orta 9 mL ekleyin, iyice hücre süspansiyon resuspend ve bir hemasitometre kullanarak hücreleri saymak.</li>
	<li>6 x 104 hücre/ml MEM içinde hücre süspansiyon yoğunluğunu ayarlayın.</li>
	<li>Hücre süspansiyon, 100 µL denetimine ekleyin ve bir steril, düz dipli, doku kültürü tedavi 96-şey plaka (şekil 1A) kuyu test.</li>
	<li>MEM kültür ortamının 100 µL 96-şey plaka (şekil 1A) boş wells için ekleyin.</li>
	<li>96-şey plaka 24 h 37 ° C'de kuluçkaya. Not: 24 h kuluçka HeLa hücreleri endojen HDAC etkinlik için test etmek için uygun bir zaman değil. Farklı hücre tipleri bilinmeyen HDAC aktivite ile kontrol hücreleri timecourse değerlendirilmesi gerekebilir.</li>
</ol>
2. hücre tedavisi ile Test bileşikleri ve HDAC yüzeylerde
<ol>
	<li>96-şey plaka kuyulardan orta Aspire edin.</li>
	<li>2 test bileşikler x 25 µL eklemek (örneğin, trichostatin A, MS275 ve tubastatin A) kuyu test etmek için MEM içinde. MEM 25 µL ve boş wells için ekleyin. Not: Test bileşiklerin en az 5 son test konsantrasyonları (başına iyi bir konsantrasyon) bir dizi sağlamak için ayarlanacak bölgedir. Bileşik testinde DMSO son konsantrasyonu % 0.5 geçmemelidir ve boşluklar ve denetimler de dahil olmak üzere her şey aynı olmalıdır. İyi ücret 50 µL son kuluçka birimdir. Trichostatin A 500 1.95 için arasında değişen 5 konsantrasyonlarda test nM (1:4 seyreltme). MS275 ve tubastatin A test 8.000 6.17 için arasında değişen 5 konsantrasyonlarda nM (1:6 seyreltme). Her test bileşik için istenen son test konsantrasyon her bireysel kullanıcı tarafından bir doz-yanıt eğrisi oluşturma tarafından tespit edilmelidir.</li>
	<li>Substrat karışımı (MAL, MOCPAC, 42 µM ve MEM BATCP 42 µM 42 µM) 25 µL kontrol ve test wells için ekleyin. MEM 25 µL boş wells için ekleyin. Not: Her substrat son konsantrasyonu 21 µM. olduğunu MOCPAC ve BATCP sırasıyla HDAC1 ve HDAC6 etkinlik arasında ayrım (şekil 1B). Hiçbir etkinlik ile bu yüzeylerde gözlenen zaman MAL kullanımı başka bir HDAC izoformu karşı faaliyetlerin tanımlaması için yol açması olası.</li>
	<li>96-şey plaka için oksijen %5 8 h 37 ° C'de kuluçkaya CO2 kuluçka makinesi. Not: Bu kuluçka süresi HeLa hücreleri için uygun ve endojen HDAC sentetik yüzeyler ile etkileşimine izin verir. Farklı hücre hatları kuluçka zaman ve/veya substrat konsantrasyonu ayarlamalar yapılması gerekebilir.</li>
</ol>
3. hücre lizis ve UHPLC-ESI-MS/MS için numune hazırlama
Dikkat: Son derece yanıcı ve yenmesi veya inhalasyon zehirli organik kimyasallar örnek hazırlık adımları kullanın. Eldiven ve koruyucu gözlük gibi uygun kişisel koruma giymek.
<ol>
	<li>10 µL (10 x RIPA arabelleğinden seyreltilmiş) 6 x RIPA arabelleği her de 96-şey plaka proteaz inhibitörü ile takıma ekleyin.</li>
	<li>Her şey soğuk Asetonitril 160 µL ekleyerek reaksiyonu durdurmak ve yukarı ve aşağı pipetting tarafından karıştırın. Not: Bu adım önce-20 ° C'de Asetonitril tutun.</li>
	<li>Plaka 10 dk-80 ° C dondurucuya koyun.</li>
	<li>Ve her şey içeriğinin 220 µL bir non-steril, konik-alt (V-alt) 96-şey plaka için transfer dondurucudan plaka çıkarın. Plaka 5000 x g ve 4 ° C'de 10 dakika santrifüj kapasitesi. Not: Proteinler ve tuzları kaldırma bu adımı amaçlamaktadır. Gerekirse, çözümler bireysel konik santrifüj tüpleri Santrifüjü önce transfer veya 96-şey, 0.65 µm PVDF filtre plakaları bir grup üzerinden filtre. Çökelti başarılı kaldırılması UHPLC analiz önce gereklidir.</li>
	<li>UHPLC sistemi ile uyumlu bir 96-şey plaka içine süpernatant (veya filtrate) 200 µL transfer ve plaka sealer kullanarak soyulabilen, termo pişirme folyo folyo ile kapatın. Not: granül supernatants kaldırırken pipetting kaçının. UHPLC analiz hemen gerçekleştirilemez, plaka en fazla 24 h 4 ° C'de analiz kadar saklanabilir.</li>
</ol>
4. UHPLC/MS-MS analizi
<ol>
	<li>UHPLC sistem mobil faz sistemiyle (%95 A:5% B) doldurarak hazırlayın: A: %0,1 formik asit içeren H2O, HPLC-grade, (1 L hazırlamak) B: Asetonitril, HPLC-grade, %0,1 formik asit içeren (1 L hazırlamak). Dikkat: Mobil faz son derece yanıcı ve yenmesi veya inhalasyon zehirli organik kimyasallar içerir. Eldiven ve koruyucu gözlük gibi uygun kişisel koruma giymek.</li>
	<li>96-şey plaka plaka sahibi içeren örnek Yöneticisi yerleştirin. Tablo 1' de sağlanan UHPLC-ESI-MS/MS koşullara göre örneklerini çalıştırma. Not: Analitik 96-şey plaka her sütun bir denetim iyi ve bir boş iyi ( şekil 1Aplaka düzeninde tasvir) olmalıdır. MS kaynak tutarlı arka plan sağlayan olup olmadığını kontrol etmek için boşlukları varken % 100 test edilmiş hücre kültürünü enzimatik aktivite temsilcisi denetimleridir. Verilen boş olarak algılanan alışılmadık MS doruklarına MS duyarlılık değişiklikleri önlemek için araştırılmalıdır.</li>
</ol>
5. veri analizi
<ol>
	<li>Her yüzey ve deacetylated ürün pik alanı uygun bir UHPLC entegrasyon yazılımı ile entegre. Not: otomatik entegrasyon parametreleri düzeltme yöntemi (ortalama, pencere boyutu 3 ve 2, örneğin pürüzsüz sayısı) kullanın. Tablo 1' de sağlanan Kromatografik koşullar kullanarak, elüsyon dMAL (5,8 dk), dBATCP (6.0 dk), dMOCPAC (6.2 dk), MAL (7.6 dk), MOCPAC (8,9 dk) ve BATCP (9,8 dk), şekil 1 ciçinde tasvir sırasıdır.</li>
	<li>Her substrat (acetylated deacetylated?) / her Kromatografik (test ve denetim örnekleri) en yüksek alan oranını hesaplamak.</li>
	<li>Her test örneği yüzde HDAC inhibisyonu Hesapla: 
	<img alt="Equation" src="/files/ftp_upload/55878/55878eq1.jpg"> Not: HDAC1 inhibisyon dMOCPAC/MOCPAC pik alanı oranı kullanılarak hesaplanır; HDAC6 inhibisyon dBATCP/BATCP ile hesaplanır. Oranı dMAL/MAL genel HDAC inhibisyon ifade etmek için kullanılabilir.</li>
</ol>

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J., Velena, A., Lee, S., Sippl, W., Jung, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-Based+Assays+for+HDAC+Inhibitor+Hit+Validation.">Cell-Based Assays for HDAC Inhibitor Hit Validation.</a> Epigenetic Targets in Drug Discovery. , (2010).</li><li>Milli, A., 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=Proteomic+analysis+of+cellular+response+to+novel+proapoptotic+agents+related+to+atypical+retinoids+in+human+IGROV-1+ovarian+carcinoma+cells.">Proteomic analysis of cellular response to novel proapoptotic agents related to atypical retinoids in human IGROV-1 ovarian carcinoma cells.</a> J Proteome Res. 10 (3), 1191-1207 (2011).</li><li>Zwick, V., 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=Synthesis+of+a+selective+HDAC6+inhibitor+active+in+neuroblasts.">Synthesis of a selective HDAC6 inhibitor active in neuroblasts.</a> Bioorg Med Chem Lett. 26 (20), 4955-4959 (2016).</li><li>Ciossek, T., Julius, H., Wieland, H., Maier, T., Beckers, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+homogeneous+cellular+histone+deacetylase+assay+suitable+for+compound+profiling+and+robotic+screening.">A homogeneous cellular histone deacetylase assay suitable for compound profiling and robotic screening.</a> Anal Biochem. 372 (1), 72-81 (2008).</li><li>Heltweg, B., Dequiedt, F., Marshall, B. 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Yazarlar ifşa gerek yok.

HDAC inhibisyon geçerli odaklı bir kanser tedavisi21HDAC6-selektif inhibitörü ilaç keşif sıcak bir konudur. HDAC seçicilik genellikle bireysel HDAC izoformlarının21doğru inhibitör etki gücüne belirlemek niyetinde yüksek üretilen iş, boş hücre deneyleri bir dizi tarafından değerlendirilir. Ancak, bir inhibitörü selectivity ayrıca canlı hücrelerde Histon gibi endojen protein yüzeylerde asetilasyon durumunu değerlendirerek onaylandığı takdirde (i...

HDACs enzim Histon Kromatin yapısı içinde deacetylate mümkün bir aileye ait. Onlar da diğer protein yüzeylerde sitozol içinde ve çeşitli hücre bölmeleri içinde bulunurlar. 18 HDAC izoformlarının toplam defa tespit edilmiştir ve transkripsiyon faktörleri ve gen ifadesinin yanı sıra hücre sinyallemesi düzenlenmesi de dahil olmak üzere birkaç hücre mekanizmaları ile ilgili ve1,2,3,4,5,6,7taşıma. HDAC katalitik inhibitörleri kanser tedavisi için potansiyel terapötik ilaç olarak ortaya çıkmıştır. Şu anda FDA tarafından onaylanmış çoğu HDAC inhibitörleri T hücreli Lenfoma ve multiple Miyelom tedavisi için ve non-spesifik HDAC inhibitörleri, vorinostat (SAHA), belinostat ve panobinostat8,9gibi. Ancak, yan etkileri bir dizi pan-inhibitörleri ile ilişkili olmuştur ve izoformu özgü küçük moleküller için arama tıbbi kimya ve ilaç bulma gelen sıcak bir konudur. Buna göre sınıf ı (HDAC1-3 ve HDAC8) selektif inhibitörü romidepsin olduğunu bir onaylanmış durumdaki ilaç10, HDAC6 özgü inhibitörleri şu anda klinik çalışmalarda, Multipl Miyelom11,12,13,14,15artan tedavi potansiyele sahip altında iken.
Deneyleri HDAC karakterize etmek için eleme inhibitörleri (tek izoformu, nükleer özü veya hücre lysate) enzimatik kaynağı olan bir HDAC substrat kuluçka temel alır. Belgili tanımlık substrate genellikle N-(4-methyl-7-aminocoumarinyl)-Nα-(t-butoxycarbonyl)-Nω-acetyllysineamide (MAL)16gibi bir yarilabilir fluorophore (Örneğin, coumarin) birleştiğinde bir asetil lizin kalıntı içeren bir küçük peptid sırası'dır. İzoformu özel etkinlikler arasında ayırt etmek için her izoformu içeren ayrı boş hücre deneyleri gerekli olan ve canlı hücreler gerçek izoformu etkinliğinde yansıtmıyor. İzoformu özel yüzeylerde Benzil gibi piyasada (S)-[1-(4-methyl-2-oxo-2H-chromen-7-ylcarbamoyl)-5-propionylaminopentyl]carbamate (MOCPAC, HDAC1 belirli substrat) ve (S)-[5-acetylamino-1-(2-oxo-4-trifluoromethyl-2H-chromen-7-ylcarbamoyl)pentyl]carbamic asit tert-butil ester (BATCP, HDAC6 belirli substrat) (şekil 1B). Onlar aynı yarilabilir fluorophore ayı verilen bu ancak, MAL içeren bir çok substrat karışımı, MOCPAC ve canlı hücreler için verilen BATCP deacetylated ürünlerin tek tespiti Fluorometrik ölçüm tarafından izin vermeyeceğim.
Burada açıklanan yöntemi algılama ve her yüzey ve deacetylated ürün UHPLC-ESI-MS/MS analiz17tarafından takip bir çok maddeyi tahlil kullanarak HeLa hücreleri içinde göreli miktar olanak sağlar. Bir HDAC tahlil doğrudan kimlik HDAC inhibitör etkinliği ve özgüllük test bileşikler endojen HDACs etkinleştirmek için HeLa hücreleri üzerinde yapılan. HDAC1 ve HDAC6, aynı anda değerlendirilir bir odak vardır. Bir tek kuluçka tahlil enzimatik Bu ölçümlerde elde etmek için non-spesifik ve belirli HDAC yüzeylerde karışımı bir 96-şey plaka üzerinde kaplama tedavi ve tedavi edilmezse HeLa hücreleri eklenir. Bir kuluçka adım hücreleri yüzeylerde ve ayrılır ve bir UHPLC-MS (şekil 1 c) yöntemiyle tespit onların anılan sıraya göre tepki ürünler serbest bırakmak için lysed. MAL, MOCPAC ve BATCP yüzeylerde deacetylated ürünleri deacetylated MAL (dMAL), deacetylated MOCPAC (dMOCPAC) ve deacetylated BATCP (dBATCP), sırasıyla vardır. Doz-yanıt eğrileri aktif bileşikler ile inşa edilebilir.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55878/55878fig1.jpg"> 
Şekil 1: HDAC1 ve HDAC6 özel inhibitörleri birden çok yüzeylerde UHPLC-MS analizi tarafından tanımlamak hücre tabanlı HDAC tahlil için genel düzeni. Tipik 96-şey plaka tedavi (test bileşikleri) içeren bir(a)düzeni ve tedavi edilmezse (kontrol) HeLa hücreleri yanı sıra boş hücre boşlukları. (B) kimyasal yapısı yüzeylerde endojen HDACs tarafından deacetylated için bir karışım (21 her µM) eklendi. (C) eklenen yüzeylerde (MAL, MOCPAC ve BATCP) ve deacetylated ürünlerini doruklarına gösterilen tipik UHPLC-MS Kromatografik (dMAL, dMOCPAC ve dBACTP, sırasıyla). <a href="http://ecsource.jove.com/files/ftp_upload/55878/55878fig1large.jpg" target="_blank">Bu rakam daha büyük bir versiyonunu görüntülemek için buraya tıklayınız.</a>

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">BATCP</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:152px;">B4061</td>
			<td style="width:273px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MAL</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td>SCP0168</td>
			<td>Synonym: BOC-Ac-Lys-AMC&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">MOCPAC</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td>M2195</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">MS275</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:152px;">EPS002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Trichostatin A</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:152px;">T8552</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Tubastatin A</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:152px;">SML0044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Acetonitrile, HPLC grade</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:152px;">10660131</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Formic acid, LC/MS grade</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:152px;">10596814</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:271px;">H2O, HPLC grade</td>
			<td style="width:191px;"></td>
			<td style="width:152px;"></td>
			<td>distilled H2O filtered through a Milli-Q purification system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">HeLa cells</td>
			<td style="width:191px;">ATCC</td>
			<td style="width:152px;">ATCC CRM-CCL-2</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell dissociation reagent TrypLE Express</td>
			<td style="width:191px;">ThermoFisher Scientific</td>
			<td style="width:152px;">12604013</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">DMSO, cell culture grade</td>
			<td style="width:191px;">Applichem</td>
			<td style="width:152px;">146463</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">DPBS</td>
			<td style="width:191px;">ThermoFisher Scientific</td>
			<td>14190144</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Fetal bovine serum&#160;</td>
			<td style="width:191px;">Biowest</td>
			<td>S1810</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">MEM</td>
			<td style="width:191px;">ThermoFisher Scientific</td>
			<td>22561021</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-Streptomycin</td>
			<td style="width:191px;">Bioconcept</td>
			<td>4-01F00-H&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10X RIPA buffer</td>
			<td style="width:191px;">Abcam</td>
			<td style="width:152px;">ab156034</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">SigmaFast protease inhibitor tablets</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:152px;">S8820</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">96-well plates, sterile, flat-bottom, tissue culture treated Corning</td>
			<td style="width:191px;">VWR</td>
			<td style="width:152px;">29442-058</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">96-well plates, non-sterile, V-bottom Corning</td>
			<td style="width:191px;">VWR</td>
			<td style="width:152px;">29442-404</td>
			<td>used in the centrifugation step</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">96-well plate, conical bottom, Nunc</td>
			<td style="width:191px;">ThermoFisher Scientific</td>
			<td style="width:152px;">249944</td>
			<td>compatible with the Acquity UHPLC system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">T75&#160; cell culture flasks Corning</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:152px;">CLS430641&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Peelable heat sealing foil&#160;</td>
			<td style="width:191px;">Waters</td>
			<td style="width:152px;">186002789</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Acquity UPLC system</td>
			<td style="width:191px;">Waters</td>
			<td style="width:152px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Eppendorf Centrifuge 5810 R</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:152px;">05-413-323</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Integration software: MassLynx V4.1</td>
			<td style="width:191px;">Waters</td>
			<td style="width:152px;"></td>
			<td>Catalog number not available</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Combi thermo-sealer SP-0669/240</td>
			<td style="width:191px;">Waters</td>
			<td style="width:152px;"></td>
			<td>Catalog number not available</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:271px;">Quattro micro API Tandem Quadrupole System</td>
			<td style="width:191px;">Waters</td>
			<td style="width:152px;"></td>
			<td>Catalog number not available</td>
		</tr>
	</tbody>
</table>

simultaneous measurement of hdac1 and hdac6 activity in hela cells using uhplc-ms

Yeni Histon deacetylase (HDAC) inhibitörleri ilaç keşif ilgi artan bir arayıştır. İzoformu seçicilik romidepsin, bir sınıf onay beri spot oldu ben HDAC inhibitörü kanser tedavisi ve HDAC6 özgü yavaşlatıcılar, Multipl Miyelom için klinik incelenmesi için. Mevcut yöntemi HDAC1 ve HDAC6 hücrelerde test bileşikler inhibitör etkinliğini belirlemek için kullanılır. İzoformu etkinlik ultra yüksek performanslı sıvı Kromatografi-Kütle spektrometresi (UHPLC-MS) analizi belirli yüzeylerde tedavi ve tedavi edilmezse HeLa hücreleri ile inkübe ölçülür. Yöntem boş hücre biyokimyasal testleri izole izoformlarının üzerinde yürütülen aksine hücre ortamında endojen HDAC etkinliği yansıtan bir avantaja sahiptir. Ayrıca, sentetik yüzeylerde miktar üzerinde temel aldığından, yöntem antikor tanıma endojen acetylated proteinlerin gerektirmez. Birden fazla hücre satır ve otomatik bir işlemin kolayca uyarlanabilir. Yöntem zaten neuroblasts içinde HDAC6-seçici bileşikler bulmada faydalı olmuştur. Temsilcisi sonuçları are göstermek burada standart HDAC inhibitörleri trichostatin ile bir (non-spesifik), MS275 (HDAC1 özel) ve tubastatin (HDAC6 özel) kullanarak HeLa hücreleri.

Non-selektif ve selektif inhibitörü HDAC1 için tanımlayıcı uygulamanın yönteminin göstermek için (sınıf ı) ve HDAC6 (sınıf IIB), HeLa hücreleri bilinen standart bileşenleri ile tedavi: trichostatin A (non-selektif HDAC1 ve HDAC6 arasında)18, MS275 (HDAC1-selektif inhibitörü)19ve tubastatin (HDAC6-selektif inhibitörü)20. Mevcut yöntem (şekil 1) kullanarak, ŞA5...

Watch this Scientific Journal Video about Simultaneous Measurement of HDAC1 and HDAC6 Activity in HeLa Cells Using UHPLC-MS at JoVE.com

Simultaneous Measurement of HDAC1 and HDAC6 Activity in HeLa Cells Using UHPLC-MS

Mevcut Yöntem birden çok yüzeylerde UHPLC-MS analizi tarafından Histon uyku (HDAC) HeLa hücreleri izoformu özgü inhibitörleri tanımlanmasını sağlar. Bu yaşam HDAC1 ve HDAC6 etkinliği yansıtmak üzere geliştirilen bir antikor-Alerjik yöntemdir tek-izoformu boş hücre deneyleri aksine hücre çevre.

Eş zamanlı ölçüm HDAC1 ve HDAC6 faaliyete HeLa hücreleri kullanarak UHPLC-MS

The search for new histone deacetylase (HDAC) inhibitors is of increasing interest in drug discovery. Isoform selectivity has been in the spotlight since ...

simultaneous-measurement-hdac1-hdac6-activity-hela-cells-using-uhplc

Universidad de Cartagena UDEC

Research

JoVE Journal

Chemistry

8.4K Görüntüleme.  Universités des Geneva–Lausanne (EPGL). Mevcut Yöntem birden çok yüzeylerde UHPLC-MS analizi tarafından Histon uyku (HDAC) HeLa hücreleri izoformu özgü inhibitörleri tanımlanmasını sağlar. Bu yaşam HDAC1 ve HDAC6 etkinliği yansıtmak üzere geliştirilen bir antikor-Alerjik yöntemdir tek-izoformu boş hücre deneyleri aksine hücre çevre.

Video: Simultaneous Measurement of HDAC1 and HDAC6 Activity in HeLa Cells Using UHPLC-MS

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry (UPLC-HRMS)

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first separated into polar and non-polar fractions by a liquid-liquid phase extraction, and partially purified to facilitate downstream analysis. Both aqueous (polar metabolites) and organic (non-polar metabolites) phases of the initial extraction are processed to survey a broad range of metabolites. Metabolites are separated by different liquid chromatography methods based upon their partition properties. In this method, we present microflow ultra-performance (UP)LC methods, but the protocol is scalable to higher flows and lower pressures. Introduction into the mass spectrometer can be through either general or compound optimized source conditions. Detection of a broad range of ions is carried out in full scan mode in both positive and negative mode over a broad m/z range using high resolution on a recently calibrated instrument. Label-free differential analysis is carried out on bioinformatics platforms. Applications of this approach include metabolic pathway screening, biomarker discovery, and drug development.

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry UPLC-HRMS

Untargeted metabolomics provides a hypothesis generating snapshot of a metabolic profile. This protocol will demonstrate the extraction and analysis of metabolites from cells, serum, or tissue. A range of metabolites are surveyed using liquid-liquid phase extraction, microflow ultraperformance liquid chromatography/high-resolution mass spectrometry (UPLC-HRMS) coupled to differential analysis software.

Ultra Performanslı Sıvı Kromatografi-Yüksek Çözünürlüklü Kütle Spektrometre (UPLC-HRMS) kullanarak Biyolojik Kaynaklar gelen hedefsiz Metabolomik

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first ...

Kimya

Steady-state, Pre-steady-state, and Single-turnover Kinetic Measurement for DNA Glycosylase Activity

Human 8-oxoguanine DNA glycosylase (OGG1) excises the mutagenic oxidative DNA lesion 8-oxo-7,8-dihydroguanine (8-oxoG) from DNA. Kinetic characterization of OGG1 is undertaken to measure the rates of 8-oxoG excision and product release. When the OGG1 concentration is lower than substrate DNA, time courses of product formation are biphasic; a rapid exponential phase (i.e. burst) of product formation is followed by a linear steady-state phase. The initial burst of product formation corresponds to the concentration of enzyme properly engaged on the substrate, and the burst amplitude depends on the concentration of enzyme. The first-order rate constant of the burst corresponds to the intrinsic rate of 8-oxoG excision and the slower steady-state rate measures the rate of product release (product DNA dissociation rate constant, koff). Here, we describe steady-state, pre-steady-state, and single-turnover approaches to isolate and measure specific steps during OGG1 catalytic cycling. A fluorescent labeled lesion-containing oligonucleotide and purified OGG1 are used to facilitate precise kinetic measurements. Since low enzyme concentrations are used to make steady-state measurements, manual mixing of reagents and quenching of the reaction can be performed to ascertain the steady-state rate (koff). Additionally, extrapolation of the steady-state rate to a point on the ordinate at zero time indicates that a burst of product formation occurred during the first turnover (i.e. y-intercept is positive). The first-order rate constant of the exponential burst phase can be measured using a rapid mixing and quenching technique that examines the amount of product formed at short time intervals (&lt;1 sec) before the steady-state phase and corresponds to the rate of 8-oxoG excision (i.e. chemistry). The chemical step can also be measured using a single-turnover approach where catalytic cycling is prevented by saturating substrate DNA with enzyme (E&gt;S). These approaches can measure elementary rate constants that influence the efficiency of removal of a DNA lesion.

Time courses for the glycosylase activity of 8-oxoguanine DNA glycosylase are biphasic exhibiting a burst of product formation and a linear steady-state phase. Utilizing quench-flow techniques, the burst and the steady-state rates can be measured, which correspond to excision of 8-oxoguanine and release of the glycosylase from the product DNA, respectively.

DNA glikosilaz Faaliyet için kararlı durum, Ön kararlı durum ve Tek cirosu Kinetik Ölçüm

Human 8-oxoguanine DNA glycosylase (OGG1) excises the mutagenic oxidative DNA lesion 8-oxo-7,8-dihydroguanine (8-oxoG) from DNA. Kinetic characterization ...

A Simple and Rapid Protocol for Measuring Neutral Lipids in Algal Cells Using Fluorescence

Algae are considered excellent candidates for renewable fuel sources due to their natural lipid storage capabilities. Robust monitoring of algal fermentation processes and screening for new oil-rich strains requires a fast and reliable protocol for determination of intracellular lipid content. Current practices rely largely on gravimetric methods to determine oil content, techniques developed decades ago that are time consuming and require large sample volumes. In this paper, Nile Red, a fluorescent dye that has been used to identify the presence of lipid bodies in numerous types of organisms, is incorporated into a simple, fast, and reliable protocol for measuring the neutral lipid content of Auxenochlorella protothecoides, a green alga. The method uses ethanol, a relatively mild solvent, to permeabilize the cell membrane before staining and a 96 well micro-plate to increase sample capacity during fluorescence intensity measurements. It has been designed with the specific application of monitoring bioprocess performance. Previously dried samples or live samples from a growing culture can be used in the assay.

A simple protocol to determine the neutral lipid content of algal cells using a Nile Red staining procedure is described. This time-saving technique offers an alternative to traditional gravimetric-based lipid quantification protocols. It has been designed for the specific application of monitoring bioprocess performance.

Floresan kullanma Alg hücrelerinde nötral Lipidler Ölçüm Basit ve Hızlı Protokolü

Algae are considered excellent candidates for renewable fuel sources due to their natural lipid storage capabilities. Robust monitoring of algal ...

HPLC Measurement of the DNA Oxidation Biomarker, 8-oxo-7,8-dihydro-2&#8217;-deoxyguanosine, in Cultured Cells and Animal Tissues

Oxidative stress is associated with many physiological and pathological processes, as well as xenobiotic metabolism, leading to the oxidation of biomacromolecules, including DNA. Therefore, efficient detection of DNA oxidation is important for a variety of research disciplines, including medicine and toxicology. A common biomarker of oxidatively damaged DNA is 8-oxo-7,8-dihydro-2&#39;-deoxyguanosine (8-oxo-dGuo; often erroneously referred to as 8-hydroxy-2&#39;-deoxyguanosine (8-OH-dGuo or 8-oxo-dG)). Several protocols for 8-oxo-dGuo measurement by high pressure liquid chromatography with electrochemical detection (HPLC-ED) have been described. However, these were mainly applied to purified DNA treated with pro-oxidants. In addition, due to methodological differences between laboratories, mainly due to differences in analytical equipment, the adoption of published methods for detection of 8-oxo-dGuo by HPLC-ED requires careful optimization by each laboratory. A comprehensive protocol, describing such an optimization process, is lacking. Here, a detailed protocol is described for the detection of 8-oxo-dGuo by HPLC-ED, in DNA from cultured cells or animal tissues. It illustrates how DNA sample preparation can be easily and rapidly optimized to minimize undesirable DNA oxidation that can occur during sample preparation. This protocol shows how to detect 8-oxo-dGuo in cultured human alveolar adenocarcinoma cells (i.e., A549 cells) treated with the oxidizing agent KBrO3, and from the spleen of mice exposed to the polycyclic aromatic hydrocarbon dibenzo(def,p)chrysene (DBC, formerly known as dibenzo(a,l)pyrene, DalP). Overall, this work illustrates how an HPLC-ED methodology can be readily optimized for the detection of 8-oxo-dGuo in biological samples.

The goal of this protocol is the detection of the DNA oxidation marker, 8-oxo-7,8-dihydro-2&#39;-deoxyguanosine (8-oxo-dGuo) by HPLC-ED, in DNA from cultured cells or animal tissues.

DNA, Oksidasyon Biomarker HPLC ölçümü, 8-okso-7,8-dihidro-2&#39;-deoksiganozin, Kültürlenmiş Hücrelerde ve hayvansal dokular

Oxidative stress is associated with many physiological and pathological processes, as well as xenobiotic metabolism, leading to the oxidation of ...

Dynamic Electrochemical Measurement of Chloride Ions

This protocol describes the dynamic measurement of chloride ions using the transition time of a silver silver chloride (Ag/AgCl) electrode. Silver silver chloride electrode is used extensively for potentiometric measurement of chloride ions concentration in electrolyte. In this measurement, long-term and continuous monitoring is limited due to the inherent drift and the requirement of a stable reference electrode. We utilized the chronopotentiometric approach to minimize drift and avoid the use of a conventional reference electrode. A galvanostatic pulse is applied to an Ag/AgCl electrode which initiates a faradic reaction depleting the Cl&#713; ions near the electrode surface. The transition time, which is the time to completely deplete the ions near the electrode surface, is a function of the ion concentration, given by the Nernst equation. The square root of the transition time is in linear relation to the chloride ion concentration. Drift of the response over two weeks is negligible (59 &#181;M/day) when measuring 1 mM [Cl&#713;]using a current pulse of 10 Am-2. This is a dynamic measurement where the moment of transition time determines the response and thus is independent of the absolute potential. Any metal wire can be used as a pseudo-reference electrode, making this approach feasible for long-term measurement inside concrete structures.

Dynamic measurement of chloride ions is presented. Transition time of an Ag/AgCl electrode, during a chronopotentiometric technique, can give the concentration of chloride ions in electrolyte. This method does not require a stable conventional reference electrode.

Klorür iyonlarının Dinamik Elektrokimyasal Ölçüm

This protocol describes the dynamic measurement of chloride ions using the transition time of a silver silver chloride (Ag/AgCl) electrode. Silver silver ...

Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical (Methylation) and Physical (Mass Spectrometry, Nuclear Magnetic Resonance) Techniques

This protocol describes the specific techniques used for the characterization of reducing end (RE) and internal region glycosyl sequence(s) of heteroxylans. De-starched wheat endosperm cell walls were isolated as an alcohol-insoluble residue (AIR)1 and sequentially extracted with water (W-sol Fr) and 1 M KOH containing 1% NaBH4 (KOH-sol Fr) as described by Ratnayake et al. (2014)2. Two different approaches (see summary in Figure 1) are adopted. In the first, intact W-sol AXs are treated with 2AB to tag the original RE backbone chain sugar residue and then treated with an endoxylanase to generate a mixture of 2AB-labelled RE and internal region reducing oligosaccharides, respectively. In a second approach, the KOH-sol Fr is hydrolyzed with endoxylanase to first generate a mixture of oligosaccharides which are subsequently labelled with 2AB. The enzymically released ((un)tagged) oligosaccharides from both W- and KOH-sol Frs are then methylated and the detailed structural analysis of both the native and methylated oligosaccharides is performed using a combination of MALDI-TOF-MS, RP-HPLC-ESI-QTOF-MS and ESI-MSn. Endoxylanase digested KOH-sol AXs are also characterized by nuclear magnetic resonance (NMR) that also provides information on the anomeric configuration. These techniques can be applied to other classes of polysaccharides using the appropriate endo-hydrolases.

Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical Methylation and Physical Mass Spectrometry, Nuclear Magnetic Resonance Techniques

This protocol describes the specific techniques used for the structural characterization of reducing end (RE) and internal region glycosyl sequence(s) of heteroxylans by tagging the RE with 2 aminobenzamide prior to enzymatic (endoxylanase) hydrolysis and then analysis of the resultant oligosaccharides using mass spectrometry (MS) and nuclear magnetic resonance (NMR).

Enzimatik, kimyasal (Metilasyon) ve Fiziksel (Kütle Spektrometre, Nükleer Manyetik Rezonans) Teknikleri Kullanarak Bitki Duvar heteroksilanlar sıralanması

This protocol describes the specific techniques used for the characterization of reducing end (RE) and internal region glycosyl sequence(s) of ...

Fabrication and Testing of Photonic Thermometers

In recent years, a push for developing novel silicon photonic devices for telecommunications has generated a vast knowledge base that is now being leveraged for developing sophisticated photonic sensors. Silicon photonic sensors seek to exploit the strong confinement of light in nano-waveguides to transduce changes in physical state to changes in resonance frequency. In the case of thermometry, the thermo-optic coefficient, i.e., changes in refractive index due to temperature, causes the resonant frequency of the photonic device such as a Bragg grating to drift with temperature. We are developing a suite of photonic devices that leverage recent advances in telecom compatible light sources to fabricate cost-effective photonic temperature sensors, which can be deployed in a wide variety of settings ranging from controlled laboratory conditions, to the noisy environment of a factory floor or a residence. In this manuscript, we detail our protocol for the fabrication and testing of photonic thermometers.

We describe the process of fabrication and testing of photonic thermometers.

İmalat ve fotonik termometreler test

In recent years, a push for developing novel silicon photonic devices for telecommunications has generated a vast knowledge base that is now being ...

Disentangling High Strength Copolymer Aramid Fibers to Enable the Determination of Their Mechanical Properties

Traditionally, soft body armor has been made from poly(p-phenylene terephthalamide) (PPTA) and ultra-high molecular weight polyethylene. However, to diversify the fiber choices in the United States body armor market, copolymer fibers based on the combination of 5-amino-2-(p-aminophenyl) benzimidazole (PBIA) and the more conventional PPTA were introduced. Little is known regarding the long-term stability of these fibers, but as condensation polymers, they are expected to have potential sensitivity to moisture and humidity. Therefore, characterizing the strength of the materials and understanding their vulnerability to environmental conditions is important for evaluating their use lifetime in safety applications. Ballistic resistance and other critical structural properties of these fibers are predicated on their strength. To accurately determine the strength of the individual fibers, it is necessary to disentangle them from the yarn without introducing any damage. Three aramid-based copolymer fibers were selected for the study. The fibers were washed with acetone followed by methanol to remove an organic coating that held the individual fibers in each yarn bundle together. This coating makes it difficult to separate single fibers from the yarn bundle for mechanical testing without damaging the fibers and affecting their strength. After washing, fourier transform infrared (FTIR) spectroscopy was performed on both washed and unwashed samples and the results were compared. This experiment has shown that there are no significant variations in the spectra of poly(p-phenylene-benzimidazole-terephthalamide-co-p-phenylene terephthalamide) (PBIA-co-PPTA1) and PBIA-co-PPTA3 after washing, and only a small variation in intensity for PBIA. This indicates that the acetone and methanol rinses are not adversely affecting the fibers and causing chemical degradation. Additionally, single fiber tensile testing was performed on the washed fibers to characterize their initial tensile strength and strain to failure, and compare those to other reported values. Iterative procedural development was necessary to find a successful method for performing tensile testing on these fibers.

The primary goal of the study is to develop a protocol to prepare consistent specimens for accurate mechanical testing of high strength copolymer aramid fibers, by removing a coating and disentangling the individual fiber strands without introducing significant chemical or physical degradation.

Disentangling yüksek mukavemetli kopolimer Aramid Elyaf mekanik özelliklerinin belirlenmesi etkinleştirmek için

Traditionally, soft body armor has been made from poly(p-phenylene terephthalamide) (PPTA) and ultra-high molecular weight polyethylene. However, to ...

High Resolution Physical Characterization of Single Metallic Nanoparticles

Individual molecules can be detected and characterized by measuring the degree by which they reduce the ionic current flowing through a single nanometer-scale pore. The signal is characteristic of the molecule&#39;s physicochemical properties and its interactions with the pore. We demonstrate that the nanopore formed by the bacterial protein exotoxin Staphylococcus aureus alpha hemolysin (&#945;HL) can detect polyoxometalates (POMs, anionic metal oxygen clusters), at the single molecule limit. Moreover, multiple degradation products of 12-phosphotungstic acid POM (PTA, H3PW12O40) in solution are simultaneously measured. The single molecule sensitivity of the nanopore method allows for POMs to be characterized at significantly lower concentrations than required for nuclear magnetic resonance (NMR) spectroscopy. This technique could serve as a new tool for chemists to study the molecular properties of polyoxometalates or other metallic clusters, to better understand POM synthetic processes, and possibly improve their yield. Hypothetically, the location of a given atom, or the rotation of a fragment in the molecule, and the metal oxidation state could be investigated with this method. In addition, this new technique has the advantage of allowing the real-time monitoring of molecules in solution.&#160;

Here, we present a protocol to detect discrete metal oxygen clusters, polyoxometalates (POMs), at the single molecule limit using a biological nanopore-based electronic platform. The method provides a complementary approach to traditional analytical chemistry tools used in the study of these molecules.

Tek metalik nanopartiküller yüksek çözünürlüklü fiziksel karakterizasyonu

Individual molecules can be detected and characterized by measuring the degree by which they reduce the ionic current flowing through a single ...

A New Straightforward Method for Lipophilicity (logP) Measurement using 19F NMR Spectroscopy

Fluorination has become an effective tool to optimize physicochemical properties of bioactive compounds. One of the applications of fluorine introduction is to modulate the lipophilicity of the compound. In our group, we are interested in the study of the impact of fluorination on lipophilicity of aliphatic fluorohydrins and fluorinated carbohydrates. These are not UV-active, resulting in a challenging lipophilicity determination. Here, we present a straightforward method for the measurement of lipophilicity of fluorinated compounds by 19F NMR spectroscopy. This method requires no UV-activity. Accurate solute mass, solvent and aliquot volume are also not required to be measured. Using this method, we measured the lipophilicities of a large number of fluorinated alkanols and carbohydrates.

A New Straightforward Method for Lipophilicity logP Measurement using 19F NMR Spectroscopy

A novel and straightforward variation of the shake-flask method was developed for accurate lipophilicity measurement of fluorinated compounds by 19F NMR spectroscopy.

19F NMR spektroskopi kullanarak Lipophilicity (logP) ölçüm için yeni bir basit Yöntem

Fluorination has become an effective tool to optimize physicochemical properties of bioactive compounds. One of the applications of fluorine introduction ...

Eş zamanlı ölçüm HDAC1 ve HDAC6 faaliyete HeLa hücreleri kullanarak UHPLC-MS

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