Bu çalışma kısmen Van Andel Araştırma Enstitüsü ve Ulusal Sağlık Enstitüsünden (CA181343) SBR&#39;e verilen bir araştırma ödeneği ile desteklendi

Yazarların açıklayacak bir şeyi yok.

Biyomedikal araştırma uygulamalarında antikor güvenilirliği en iyisi 46 , 47 . Bu, özellikle histamin PTM&#39;lerinin bolluğu ve dağılımını karakterize etmek için geliştirilen teknikler için anahtar araçlar olarak antikorların bulunduğu kromatin biyokimyasında geçerlidir. Burada sunulan protokol, histon PTM antikor özgünlüğünü analiz etmek için peptit mikrodizilerinin tasarım, imalat ve kullanımı için optimize edilmiş bir boru hat...

Genomik DNA, kromatin oluşturmak için ökaryotik hücre çekirdeğinin içinde histon proteinleri ile zarif bir şekilde paketlenmiştir. Kromatin tekrarlayan altbirimi, H2A, H2B, H3 ve H4 1 oktamerik bir çekirdek histon proteinleri etrafında sarılan 147 baz çift DNA&#39;dan oluşan nükleozomdur. Kromatin genel olarak gevşek biçimde paketlenmiş ökromatin ve yoğun sıkıştırılmış heterokromatin alanlar halinde düzenlenir. Kromatin sıkıştırma derecesi, protein makineleri, kopyalama, kopyalama ve onarım gibi temel DNA şablonlu işlemleri gerçekleştirmek için altta yatan DNA&#39;ya ne ölçüde erişebildiğini düzenler. Genetik erişilebilirliğin kromatin bağlamındaki kilit düzenleyicileri histon proteinlerinin yapılandırılmamış kuyruk ve çekirdek alanlarındaki 2 , 3 PTM&#39;leridir. Histon PTM&#39;leri doğrudan kromatin 4&#39;ün yapısını etkiler ve dolaylı olarak işlev görürlerh okuyucu proteinleri ve kromatin remodeling, enzimatik ve iskele aktivitelerini 5 sahip ilişkili makromoleküler kompleksler işe. Son iki on yılda histon PTM fonksiyonu üzerine yapılan çalışmalar, bu işaretlerin hücre akıbetini, organizma gelişimini ve hastalık başlatma / ilerlemesini düzenleyen önemli rol oynadığını göstermektedir. Kütle spektrometresi tabanlı proteomik teknolojideki ilerlemelerle beslenen 80&#39;den fazla farklı histon artıkları üzerinde 20&#39;den fazla benzersiz histon PTM&#39;si keşfedildi 6 . Özellikle, bu modifikasyonlar sıklıkla kombinasyon halinde ortaya çıkmakta ve &quot;histon kodu&quot; hipotezi ile tutarlı bir şekilde sayısız araştırma, histon PTM&#39;lerinin 7 , 8 , 9&#39;un spesifik kombinasyonlarının tanınması yoluyla okuyucu proteinlerinin farklı kromatin bölgelerini hedef aldığını göstermektedir. Önemli bir zorluk, işlevleri GrHistone PTM&#39;lerinin listesine bağlı olarak ve belirli histone PTM kombinasyonlarının kromatin ile ilişkili dinamik fonksiyonları nasıl düzenlediğini belirlemek için kullandı. Antikorlar, histon PTM&#39;lerinin tespiti için linç pinin reaktifleridir. Bu nedenle, kromatin biyokimya araştırmasında kullanılmak üzere 1.000&#39;den fazla histon PTM&#39;ye spesifik antikor ticari olarak geliştirilmiştir. Yüksek verimli DNA dizileme teknolojisinin hızlı gelişimi ile bu reaktifler, ChIP-seq&#39;deki bireysel araştırmacılar ve büyük ölçekli epigenomik &quot;yol haritası&quot; girişimleri ( örn. , ENCODE ve BLUEPRINT) tarafından geniş ölçüde kullanılmaktadır (kromatin immüno çökeltme, yeni nesil sıralama ) Histonun yüksek çözünürlüklü mekânsal haritalarını üretmek için boru hatları PTM dağıtım genomu genişliği 10 , 11 . Bununla birlikte, son çalışmalar histon PTM antikorlarının özgüllüğünün oldukça değişken olduğunu ve bu reaktiflerin unf<html> Hedef dışı epitop tanıma, komşu PTM&#39;ler tarafından güçlü pozitif ve negatif etki ve belirli bir kalıntı ( örn. , Mono-, di- veya tri-metillisin) 12 , 13 , 14 , 15&#39;deki modifikasyon sırasını ayırt etme zorluğu gibi avorable özellikler , 16 , 17 , 18 . Bu nedenle, bu değerli reaktiflerle üretilen verileri doğru bir şekilde yorumlamak için histon PTM&#39;ye özgü antikor reaktiflerinin titiz kalite kontrolü gereklidir. Mikroarray teknolojisi, binlerce makromoleküler etkileşimin aynı anda yüksek verimli, tekrarlanabilir ve minyatür formatta sorgulanmasını sağlar. Bu nedenle protein-DNA 19&#39;u analiz etmek için çeşitli mikrodizim platformları yaratılmıştır ,&quot;&gt; 20, protein-protein 21 ve protein-peptid etkileşimlerinin 22. Gerçekten de, histon peptid mikrodizileri yazarlar, silgi yüksek verimli profil sağlayan kromatin biyokimya araştırma için bilgi keşif platformu olarak ortaya çıktı ve histonun okuyucuları PTMS 15 adres , 23 , 24 ve ayrıca histone antikor özgünlüğünün analizi için 17 , 25. Histon peptit dizileri, kromatin ve epigenetik araştırmalarının ötesinde, sistemik lupus eritematosus ve diğer otoimmün hastalıkların teşhis / prognostik testi olarak potansiyel bir kullanıma sahiptir; kromatin otoantikorları, 27 26 oluşturulur. Burada, tasarım, imalat ve que için geliştirdiğimiz entegre bir boru hattı tanımlıyoruzHistonları ve PTM&#39;lerini tanıyan antikorlar için özgüllük profilleri üretmek için histon peptidi mikrodizileri kullandı. Boru hattı, yakın zamanda geliştirdiğimiz, mikroarray deneylerinin tasarım ve analiz aşamalarını bütünleştiren, açık kaynaklı ve interaktif bir yazılım uygulaması olan ArrayNinja tarafından kolaylaştırıldı 28 . ArrayNinja, Google Chrome&#39;da en iyi şekilde çalışır. Kısacası, streptavidin kaplı cam mikroskop lamlarında belirli bir konumda biyotinle konjuge edilmiş histone peptidlerinin bir kütüphanesinin depolanması için bir robotik temas mikrodizisi yazıcı kullanılır. Ardından diziler, antikor-epitop etkileşimlerini sorgulamak için rekabetçi ve paralel bir tahlil formunda kullanılabilir ( Şekil 1 ). Peptid kütüphanesi, tek başına PTM&#39;leri (lizin asetilasyonu, lisin / arginin metilasyonu ve serin / treonin fosforilasyonu) içeren yüzlerce benzersiz sentetik peptid ve büyük oranda proteomik veri kümelerinden türetilen ilgili kombinasyonlardan oluşur. Peptid sentezi ve geçerliliği için yöntemler<html> Başka yerlerde detaylandırılmıştır 23 . Bu dizilim platformunu kullanarak halen devam eden histon PTM antikor tarama çabalarımızdan elde edilen veriler, halka açık bir web kaynağı, Histone Antikor Özgüllilik Veritabanı (www.histoneantibodies.com) üzerinde arşivlenir. Özellikle, bu protokolün çeşitlemeleri ile imal edilen histon peptit mikroarrayleri, histon PTM okuyucu alanlarının 8 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 ve daha yakın zamanda profil histonuna aktivitesini karakterize etmek için yaygın bir şekilde kullanılmıştır PTM yazar ve silgi faaliyetleri 24 . /files/ftp_upload/55912/55912fig1.jpg &quot;/&gt; Şekil 1: Bir Histone Peptid mikroarrayinde Antikor Taramasının Kademeli Prosedürünün Çizgi Film Tasvirleri. Tanımlanmış translasyon sonrası modifikasyonları (kırmızı ve mavi daireler) barındıran biyotinlenmiş histon peptidleri, streptavidin kaplı cam üzerinde biyotin-floresein ile birlikte basılmıştır. Pozitif etkileşimler kırmızı floresans olarak görselleştirilir. <a href="http://ecsource.jove.com/files/ftp_upload/55912/55912fig1large.jpg" target="_blank">Bu rakamın daha büyük bir versiyonunu görmek için lütfen tıklayınız.</a>

<table border="0" cellpadding="0" cellspacing="0" width="746">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Printing Buffer</td>
			<td style="width:107px;">ArrayIt</td>
			<td style="width:105px;">PPB</td>
			<td style="width:281px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">BSA</td>
			<td style="width:107px;">Omnipure</td>
			<td align="right" style="width:105px;">2390</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Streptavidin-coated glass microscope slides</td>
			<td style="width:107px;">Greiner Bio-one</td>
			<td>439003-25</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">polypropylene 384 well plate</td>
			<td style="width:107px;">Greiner Bio-one</td>
			<td align="right" style="width:105px;">784201</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Biotin-fluorescein</td>
			<td style="width:107px;">Sigma</td>
			<td align="right" style="width:105px;">53608</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">contact microarray printer</td>
			<td style="width:107px;">Aushon</td>
			<td align="right" style="width:105px;">2470</td>
			<td style="width:281px;">Aushon 2470 Microarray Printer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">contact microarray printer</td>
			<td style="width:107px;">Gene Machines</td>
			<td style="width:105px;">OmniGrid 100</td>
			<td style="width:281px;">OmniGrid Microarray Printer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">PBS</td>
			<td style="width:107px;">Invitrogen</td>
			<td align="right" style="width:105px;">14190</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Blocking Buffer</td>
			<td style="width:107px;">ArrayIt</td>
			<td style="width:105px;">SBB</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Hydrophobic wax pen</td>
			<td style="width:107px;">Vector Labs</td>
			<td style="width:105px;">H-4000</td>
			<td>ImmEdge Hydrophobic Barrier PAP Pen</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Silicon Gasket</td>
			<td style="width:107px;">Grace Bio-labs</td>
			<td align="right" style="width:105px;">622511</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Hybridization Vessel</td>
			<td style="width:107px;">Thermo Scientific</td>
			<td align="right">267061</td>
			<td>or similar vessel</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Fluorescent-dye conjugated secondary antibody</td>
			<td style="width:107px;">Life Technologies</td>
			<td>A-21244</td>
			<td style="width:281px;">Alexa Fluor 647 (anti-rabbit)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Fluorescent-dye conjugated secondary antibody</td>
			<td style="width:107px;">Life Technologies</td>
			<td>A-21235</td>
			<td style="width:281px;">Alexa Fluor 647 (anti-mouse)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Wax Imprinter</td>
			<td style="width:107px;">ArrayIt</td>
			<td>MSI48</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Tween-20</td>
			<td style="width:107px;">Omnipure</td>
			<td align="right" style="width:105px;">9490</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Microarray Scanner</td>
			<td style="width:107px;">Innopsys</td>
			<td style="width:105px;">InnoScan 1100AL</td>
			<td>or equivalent microarray scanner</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">EipTitan Histone Peptide Microarray</td>
			<td style="width:107px;">Epicypher</td>
			<td align="right" style="width:105px;">112001</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">AbSurance Pro Histone Peptide Microarray</td>
			<td style="width:107px;">Millipore</td>
			<td align="right" style="width:105px;">16668</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">MODified Histone Peptide Array</td>
			<td style="width:107px;">Active Motif</td>
			<td align="right" style="width:105px;">13001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Histone Code Peptide Microarrays</td>
			<td style="width:107px;">JPT</td>
			<td style="width:105px;">His_MA_01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Wax</td>
			<td style="width:107px;">Royal Oak</td>
			<td style="width:105px;">GulfWax</td>
			<td>for wax imprinter</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Humidified Microarray Slide Hybridization Chamber</td>
			<td style="width:107px;">VWR</td>
			<td style="width:105px;">97000-284</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">High throughput microscope slide washing chamber</td>
			<td style="width:107px;">ArrayIt</td>
			<td style="width:105px;">HTW</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Microscope slide centrifuge</td>
			<td style="width:107px;">VWR</td>
			<td>93000-204</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Antibody 1</td>
			<td>Abcam</td>
			<td align="right" style="width:105px;">8898</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Antibody 2</td>
			<td style="width:107px;">Millipore</td>
			<td style="width:105px;">07-473</td>
			<td></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:255px;">Biotinylated histone peptide</td>
			<td style="width:107px;">EpiCypher</td>
			<td>12-0001</td>
			<td style="width:281px;">Example peptide. Similar peptides with various modifications are available from several commercial sources.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">ImageMagick</td>
			<td style="width:107px;"></td>
			<td style="width:105px;"></td>
			<td>https://www.imagemagick.org/script/index.php</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">ArrayNinja</td>
			<td style="width:107px;"></td>
			<td style="width:105px;"></td>
			<td>https://rothbartlab.vai.org/tools/</td>
		</tr>
	</tbody>
</table>

analysis of histone antibody specificity with peptide microarrays

Histon proteinleri üzerindeki translasyon sonrası modifikasyonlar (PTM&#39;ler), kromatin yapısını ve gen ekspresyonunu düzenleme rolleri için geniş çapta incelenmektedir. Histon PTM&#39;lerine spesifik antikorların seri olarak üretilmesi ve dağıtımı, bu işaretler üzerinde araştırmayı büyük ölçüde kolaylaştırdı. Histon PTM antikorları, birçok kromatin biyokimya uygulaması için önemli reaktifler olduğundan, doğru veri yorumlaması ve sahadaki ilerlemenin devam etmesi için antikor özgüllüğünün titiz analizi gereklidir. Bu protokol, histone antikorlarının spesifikliğini profillemek için peptit mikrodizilerinin tasarım, imalat ve kullanımı için entegre bir boru hattı tarif eder. Bu prosedürün tasarım ve analiz yönleri ArrayNinja tarafından kolaylaştırılmış ve açık kaynaklı ve etkileşimli bir yazılım paketi, son zamanlarda mikroarray baskı formatlarının uyarlanmasını hızlandırmak için geliştirdi. Bu boru hattı, piyasada bulunan ve yaygın olarak kullanılan histon PTM antibodisinin çok sayıda taranması için kullanılmıştırS ve bu deneylerden elde edilen veriler çevrimiçi ve genişletici bir Histon Antikor Özgüllilik Veritabanı aracılığıyla serbestçe elde edilebilir. Histonların ötesinde, burada açıklanan genel metod, PTM&#39;ye spesifik antikorların analizine genel olarak uygulanabilir.

Bu protokol, histone PTM antikor özgünlüğünün analizi için bir peptid mikroarray platformu tasarlamak ve üretmek için kullanılmıştır. Çekirdek ve varyant histon proteinleri 38 üzerinde bulunan PTMS bilinen kombinasyonların birçok temsil - (uzunluğu 40 kalıntıları 20) dizi 300&#39;den benzersiz peptid özellikleri bir kütüphane sorgular. Bu boru hattı, yaygın olarak kullanılan ve piyasada mevcut olan histon PTM antikorlarının taranması ...

Watch this Scientific Journal Video about Analysis of Histone Antibody Specificity with Peptide Microarrays at JoVE.com

Analysis of Histone Antibody Specificity with Peptide Microarrays

Histon Antikor Özgüllüğünün Peptit Mikroarraylerle Analizi

Bu el yazısı, histonları ve bunların translasyon sonrası modifikasyonlarını tanıyan antikorların spesifiklik profiline peptid mikroarray teknolojisinin uygulanması için yöntemleri açıklamaktadır.

Post-translational modifications (PTMs) on histone proteins are widely studied for their roles in regulating chromatin structure and gene expression. The ...

analysis-of-histone-antibody-specificity-with-peptide-microarrays

Research

JoVE Journal

Biochemistry

39.8K Görüntüleme.  Van Andel Research Institute.  Bu el yazısı, histonları ve bunların translasyon sonrası modifikasyonlarını tanıyan antikorların spesifiklik profiline peptid mikroarray teknolojisinin uygulanması için yöntemleri açıklamaktadır. 

Video: Analysis of Histone Antibody Specificity with Peptide Microarrays

Laboratory Scale Production and Purification of a Therapeutic Antibody

Ensuring the successful production of a therapeutic antibody begins early on in the development process. The first stage is vector expression of the antibody genes followed by stable transfection into a suitable cell line. The stable clones are subjected to screening in order to select those clones with desired production and growth characteristics. This is a critical albeit time-consuming step in the process. This protocol considers vector selection and sourcing of antibody sequences for the expression of a therapeutic antibody. The methods describe preparation of vector DNA for stable transfection of a suspension variant of human embryonic kidney 293 (HEK-293) cell line, using polyethylenimine (PEI). The cells are transfected as adherent cells in serum-containing media to maximize transfection efficiency, and afterwards adapted to serum-free conditions. Large scale production, setup as batch overgrow cultures is used to yield antibody protein that is purified by affinity chromatography using an automated fast protein liquid chromatography (FPLC) instrument. The antibody yields produced by this method can provide sufficient protein to begin initial characterization of the antibody. This may include in vitro assay development or physicochemical characterization to aid in the time-consuming task of clonal screening for lead candidates. This method can be transferable to the development of an expression system for the production of biosimilar antibodies.

This protocol describes the production of a therapeutic antibody in a mammalian expression system. The methods described include preparation of vector DNA, stable transfection and serum-free adaptation of a human embryonic kidney 293 cell line, set up of large scale cultures and purification using affinity chromatography.

Laboratuar ölçekli üretim ve terapötik bir antikor ile saflaştırılması

Ensuring the successful production of a therapeutic antibody begins early on in the development process. The first stage is vector expression of the ...

Biyokimya

Easy Manipulation of Architectures in Protein-based Hydrogels for Cell Culture Applications

Hydrogels are recognized as promising materials for cell culture applications due to their ability to provide highly hydrated cell environments. The field of 3D templates is rising due to the potential resemblance of those materials to the natural extracellular matrix. Protein-based hydrogels are particularly promising because they can easily be functionalized and can achieve defined structures with adjustable physicochemical properties. However, the production of macroporous 3D templates for cell culture applications using natural materials is often limited by their weaker mechanical properties compared to those of synthetic materials. Here, different methods were evaluated to produce macroporous bovine serum albumin (BSA)-based hydrogel systems, with adjustable pore sizes in the range of 10 to 70 &#181;m in radius. Furthermore, a method to generate channels in this protein-based material that are several hundred microns long was established. The different methods to produce pores, as well as the influence of pore size on material properties such as swelling ratio, pH, temperature stability, and enzymatic degradation behavior, were analyzed. Pore sizes were investigated in the native, swollen state of the hydrogels using confocal laser scanning microscopy. The feasibility for cell culture applications was evaluated using a cell-adhesive RGD peptide modification of the protein system and two model cell lines: human breast cancer cells (A549) and adenocarcinomic human alveolar basal epithelial cells (MCF7).

Different methods to manipulate three-dimensional architecture in protein-based hydrogels are evaluated here with respect to material properties. The macroporous networks are functionalized with a cell-adhesive peptide, and their feasibility in cell culture is evaluated using two different model cell lines.

Protein bazlı Hydrogels mimarilerde kolayca idare hücre kültür uygulamaları için

Hydrogels are recognized as promising materials for cell culture applications due to their ability to provide highly hydrated cell environments. The field ...

Semi-automated Biopanning of Bacterial Display Libraries for Peptide Affinity Reagent Discovery and Analysis of Resulting Isolates

Biopanning bacterial display libraries is a proven technique for peptide affinity reagent discovery for recognition of both biotic and abiotic targets. Peptide affinity reagents can be used for similar applications to antibodies, including sensing and therapeutics, but are more robust and able to perform in more extreme environments. Specific enrichment of peptide capture agents to a protein target of interest is enhanced using semi-automated sorting methods which improve binding and wash steps and therefore decrease the occurrence of false positive binders. A semi-automated sorting method is described herein for use with a commercial automated magnetic-activated cell sorting device with an unconstrained bacterial display sorting library expressing random 15-mer peptides. With slight modifications, these methods are extendable to other automated devices, other sorting libraries, and other organisms. A primary goal of this work is to provide a comprehensive methodology and expound the thought process applied in analyzing and minimizing the resulting pool of candidates. These techniques include analysis of on-cell binding using fluorescence-activated cell sorting (FACS), to assess affinity and specificity during sorting and in comparing individual candidates, and the analysis of peptide sequences to identify trends and consensus sequences for understanding and potentially improving the affinity to and specificity for the target of interest.

Biopanning bacterial display libraries is a proven technique for discovery of peptide affinity reagents, a robust alternative to antibodies. The semi-automated sorting method herein has streamlined biopanning to decrease the occurrence of false positives. Here we illustrate the thought process and techniques applied in evaluating candidates and minimizing downstream analysis.

Peptid benzeşme reaktif keşif ve elde edilen yalıtır analizi için bakteriyel ekran kütüphanelerin yarı otomatik Biopanning

Biopanning bacterial display libraries is a proven technique for peptide affinity reagent discovery for recognition of both biotic and abiotic targets. ...

The Determination of Protease Specificity in Mouse Tissue Extracts by MALDI-TOF Mass Spectrometry: Manipulating PH to Cause Specificity Changes

Proteases have several biological functions, including protein activation/inactivation and food digestion. Identifying protease specificity is important for revealing protease function. The method proposed in this study determines protease specificity by measuring the molecular weight of unique substrates using Matrix-assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) mass spectrometry. The substrates contain iminobiotin, while the cleaved site consists of amino acids, and the spacer consists of polyethylene glycol. The cleaved substrate will generate a unique molecular weight using a cleaved amino acid. One of the merits of this method is that it may be carried out in one pot using crude samples, and it is also suitable for assessing multiple samples. In this article, we describe a simple experimental method optimized with samples extracted from mouse lung tissue, including tissue extraction, placement of digestive substrates into samples, purification of digestive substrates under different pH conditions, and measurement of the substrates&#39; molecular weight using MALDI-TOF mass spectrometry. In summary, this technique allows for the identification of protease specificity in crude samples derived from tissue extracts using MALDI-TOF mass spectrometry, which may easily be scaled up for multiple sample processing.

Here, we present a protocol to determine protease specificity in crude mouse tissue extracts using MALDI-TOF spectrometry.

Proteaz özgüllük fare doku belirlenmesi MALDI-TOF Kütle spektrometresi tarafından ayıklar: PH özgüllük değişiklikler neden manipüle

Proteases have several biological functions, including protein activation/inactivation and food digestion. Identifying protease specificity is important ...

Kinase Inhibitor Screening In Self-assembled Human Protein Microarrays

The screening of kinase inhibitors is crucial for better understanding properties of a drug and for the identification of potentially new targets with clinical implications. Several methodologies have been reported to accomplish such screening. However, each has its own limitations (e.g., the screening of only ATP analogues, restriction to using purified kinase domains, significant costs associated with testing more than a few kinases at a time, and lack of flexibility in screening protein kinases with novel mutations). Here, a new protocol that overcomes some of these limitations and can be used for the unbiased screening of kinase inhibitors is presented. A strength of this method is its ability to compare the activity of kinase inhibitors across multiple proteins, either between different kinases or different variants of the same kinase. Self-assembled protein microarrays generated through the expression of protein kinases by a human-based in vitro transcription and translation system (IVTT) are employed. The proteins displayed on the microarray are active, allowing for measurement of the effects of kinase inhibitors. The following procedure describes the protocol steps in detail, from the microarray generation and screening to the data analysis.

A detailed protocol for the generation of self-assembled human protein microarrays for the screening of kinase inhibitors is presented.

Kendi kendine monte Edilen İnsan Protein Mikrodizilerinde Kiaz Inhibitörü Taraması

The screening of kinase inhibitors is crucial for better understanding properties of a drug and for the identification of potentially new targets with ...

Isolation of Histone from Sorghum Leaf Tissue for Top Down Mass Spectrometry Profiling of Potential Epigenetic Markers

Histones belong to a family of highly conserved proteins in eukaryotes. They pack DNA into nucleosomes as functional units of chromatin. Post-translational modifications (PTMs) of histones, which are highly dynamic and can be added or removed by enzymes, play critical roles in regulating gene expression. In plants, epigenetic factors, including histone PTMs, are related to their adaptive responses to the environment. Understanding the molecular mechanisms of epigenetic control can bring unprecedented opportunities for innovative bioengineering solutions. Herein, we describe a protocol to isolate the nuclei and purify histones from sorghum leaf tissue. The extracted histones can be analyzed in their intact forms by top-down mass spectrometry (MS) coupled with online reversed-phase (RP) liquid chromatography (LC). Combinations and stoichiometry of multiple PTMs on the same histone proteoform can be readily identified. In addition, histone tail clipping can be detected using the top-down LC-MS workflow, thus, yielding the global PTM profile of core histones (H4, H2A, H2B, H3). We have applied this protocol previously to profile histone PTMs from sorghum leaf tissue collected from a large-scale field study, aimed at identifying epigenetic markers of drought resistance. The protocol could potentially be adapted and optimized for chromatin immunoprecipitation-sequencing (ChIP-seq), or for studying histone PTMs in similar plants.

The protocol has been developed to effectively extract intact histones from sorghum leaf materials for profiling of histone post-translational modifications that can serve as potential epigenetic markers to aid engineering drought resistant crops.

Potansiyel Epigenetik belirteçlerin Yukarıdan Aşağıya Kütle Spektrometresi Profillemi için Sorgum Yaprak Dokusundan Histone İzolasyonu

Histones belong to a family of highly conserved proteins in eukaryotes. They pack DNA into nucleosomes as functional units of chromatin. ...

Rapid Determination of Antibody-Antigen Affinity by Mass Photometry

Measurements of the specificity and affinity of antigen-antibody interactions are critically important for medical and research applications. In this protocol, we describe the implementation of a new single-molecule technique, mass photometry (MP), for this purpose. MP is a label- and immobilization-free technique that detects and quantifies molecular masses and populations of antibodies and antigen-antibody complexes on a single-molecule level. MP analyzes the antigen-antibody sample within minutes, allowing for the precise determination of the binding affinity and simultaneously providing information on the stoichiometry and the oligomeric state of the proteins. This is a simple and straightforward technique that requires only picomole quantities of protein and no expensive consumables. The same procedure can be used to study protein-protein binding for proteins with a molecular mass larger than 50 kDa. For multivalent protein interactions, the affinities of multiple binding sites can be obtained in a single measurement. However, the single-molecule mode of measurement and the lack of labeling imposes some experimental limitations. This method gives the best results when applied to measurements of sub-micromolar interaction affinities, antigens with a molecular mass of 20 kDa or larger, and relatively pure protein samples. We also describe the procedure for performing the required fitting and calculation steps using basic data analysis software.

We describe a single-molecule approach to antigen-antibody affinity measurements using mass photometry (MP). The MP-based protocol is fast, accurate, uses a very small amount of material, and does not require protein modification.

Kitle Fotometrisi ile Antikor-Antijen Affinitesinin Hızlı Tayini

Measurements of the specificity and affinity of antigen-antibody interactions are critically important for medical and research applications. In this ...

Cell-Free Production of Proteoliposomes for Functional Analysis and Antibody Development Targeting Membrane Proteins

Membrane proteins play essential roles in a variety of cellular processes and perform vital functions. Membrane proteins are medically important in drug discovery because they are the targets of more than half of all drugs. An obstacle to conducting biochemical, biophysical, and structural studies of membrane proteins as well as antibody development has been the difficulty in producing large amounts of high-quality membrane protein with correct conformation and activity. Here we describe a &#8220;bilayer-dialysis method&#8221; using a wheat germ cell-free system, liposomes, and dialysis cups to efficiently synthesize membrane proteins and prepare purified proteoliposomes in a short time with a high success rate. Membrane proteins can be produced as much as in several milligrams, such as GPCRs, ion channels, transporters, and tetraspanins. This cell-free method contributes to reducing the time, cost and effort for preparing high-quality proteoliposomes, and provides suitable means for functional analysis of membrane proteins, drug targets screening, and antibody development.

This protocol describes an efficient cell-free method for production of high-quality proteoliposome by bilayer-dialysis method using wheat cell-free system and liposomes. This method provides suitable means for functional analysis of membrane proteins, drug targets screening, and antibody development.

Membran Proteinlerini Hedefleyen Fonksiyonel Analiz ve Antikor Gelişimi için Proteolipozomların Hücresiz Üretimi

Membrane proteins play essential roles in a variety of cellular processes and perform vital functions. Membrane proteins are medically important in drug ...

Cryo-EM and Single-Particle Analysis with Scipion

Cryo-electron microscopy has become one of the most important tools in biological research to reveal the structural information of macromolecules at near-atomic resolution. In single-particle analysis, the vitrified sample is imaged by an electron beam and the detectors at the end of the microscope column produce movies of that sample. These movies contain thousands of images of identical particles in random orientations. The data need to go through an image processing workflow with multiple steps to obtain the final 3D reconstructed volume. The goal of the image processing workflow is to identify the acquisition parameters to be able to reconstruct the specimen under study. Scipion provides all the tools to create this workflow using several image processing packages in an integrative framework, also allowing the traceability of the results. In this article the whole image processing workflow in Scipion is presented and discussed with data coming from a real test case, giving all the details necessary to go from the movies obtained by the microscope to a high resolution final 3D reconstruction. Also, the power of using consensus tools that allow combining methods, and confirming results along every step of the workflow, improving the accuracy of the obtained results, is discussed.

Single-particle analysis in cryo-electron microscopy is one of the main techniques used to determine the structure of biological ensembles at high resolution. Scipion provides the tools to create the whole pipeline to process the information acquired by the microscope and achieve a 3D reconstruction of the biological specimen.

Scipion ile Kriyo-EM ve Tek Parçacık Analizi

Cryo-electron microscopy has become one of the most important tools in biological research to reveal the structural information of macromolecules at ...

In Vitro Characterization of Histone Chaperones using Analytical, Pull-Down and Chaperoning Assays

Histone proteins associate with DNA to form the eukaryotic chromatin. The basic unit of chromatin is a nucleosome, made up of a histone octamer consisting of two copies of the core histones H2A, H2B, H3, and H4, wrapped around by the DNA. The octamer is composed of two copies of an H2A/H2B dimer and a single copy of an H3/H4 tetramer. The highly charged core histones are prone to non-specific interactions with several proteins in the cellular cytoplasm and the nucleus. Histone chaperones form a diverse class of proteins that shuttle histones from the cytoplasm into the nucleus and aid their deposition onto the DNA, thus assisting the nucleosome assembly process. Some histone chaperones are specific for either H2A/H2B or H3/H4, and some function as chaperones for both. This protocol describes how in vitro laboratory techniques such as pull-down assays, analytical size-exclusion chromatography, analytical ultra-centrifugation, and histone chaperoning assay could be used in tandem to confirm whether a given protein is functional as a histone chaperone.

In Vitro Characterization of Histone Chaperones using Analytical, Pull-Down and Chaperoning Assays

This protocol describes a battery of methods that includes analytical size-exclusion chromatography to study histone chaperone oligomerization and stability, pull-down assay to unravel histone chaperone-histone interactions, AUC to analyze the stoichiometry of the protein complexes, and histone chaperoning assay to functionally characterize a putative histone chaperone in vitro.

İn Vitro Histon Chaperonlarının Analitik, Pull-Down ve Chaperoning Testleri Kullanılarak Karakterizasyonu

Histone proteins associate with DNA to form the eukaryotic chromatin. The basic unit of chromatin is a nucleosome, made up of a histone octamer consisting ...