Questo lavoro è stato sostenuto in parte da Van Andel Research Institute e da una borsa di studio degli Istituti Nazionali di Sanità (CA181343) a SBR

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L&#39;affidabilità degli anticorpi nelle applicazioni di ricerca biomedica è fondamentale 46 , 47 . Ciò è particolarmente vero nella biochimica cromatica data la posizione degli anticorpi come strumenti chiave per la maggior parte delle tecniche sviluppate per caratterizzare l&#39;abbondanza e la distribuzione di PTM istoniche. Il protocollo qui presentato illustra una pipeline ottimizzata per la progettazione, la fabbricazione e l&#39;uso di microarray pept...

Il DNA genomico è imballato elegantemente all&#39;interno del nucleo delle cellule eucariotiche con proteine ​​dell&#39;istone per formare la cromatina. La subunità ripetizione della cromatina è il nucleosoma, che consiste di 147 coppie di basi di DNA avvolto attorno ad un nucleo octameric di proteine istoni H2A, - H2B, H3, H4 e 1. La cromatinica è ampiamente organizzata in euchromatin pienamente ricoperti e domini eterocromatina strettamente confezionati. Il grado di compattazione della cromatina regola la misura in cui i macchinari di proteine ​​possono accedere al DNA sottostante per eseguire processi fondamentali del DNA come la replica, la trascrizione e la riparazione. I regolatori chiave dell&#39;accessibilità del genoma nel contesto della cromatina sono PTM sui nodi non strutturati e nuclei delle proteine ​​istone 2 , 3 . Histon PTMs funziona direttamente influenzando la struttura della cromatina 4 e indirettamenteH l&#39;assunzione di proteine ​​di lettura e dei loro complessi macromolecolari associati con attività di ricostruzione cromatica, enzimatica e di ponteggi 5 . Studi sulla funzione istone PTM negli ultimi due decenni suggeriscono in maniera massiccia che questi segni svolgono ruoli chiave nel regolare il destino cellulare, lo sviluppo organico e l&#39;iniziazione / progressione delle malattie. Alimentata dai progressi della tecnologia proteomica basata sulla spettrometria di massa, sono stati scoperti più di 20 PTM istonici unici su più di 80 residui istoniali distinti 6 . Notevolmente, queste modifiche spesso si verificano in combinazioni e coerenti con l&#39;ipotesi del codice istone, numerosi studi suggeriscono che le proteine ​​del lettore sono destinate a regioni discrete di cromatina attraverso il riconoscimento di combinazioni specifiche di istone PTMs 7 , 8 , 9 . Una sfida fondamentale in avanti sarà attribuire funzioni al grGrazie alla lista degli istoni PTMs e per determinare come combinazioni specifiche di PTM istoni orchestrino le funzioni dinamiche associate alla cromatina. Gli anticorpi sono i reagenti lynchpin per la rilevazione di PTM istonico. In quanto tale, più di 1.000 anticorpi specifici PTM-istone sono stati commercialmente sviluppati per l&#39;uso nella ricerca biochimica cromatina. Con il rapido sviluppo di high-throughput tecnologia di sequenziamento del DNA, questi reagenti sono stati ampiamente utilizzati dai singoli ricercatori e epigenomics grandi &quot;calendario&quot; iniziative (ad esempio, codificare e progetto), in ChIP-seq (cromatina immunoprecipitazione accoppiato con sequenziamento di prossima generazione ) Per generare mappe spaziali ad alta risoluzione della distribuzione istone PTM a livello genomico 10 , 11 . Tuttavia, recenti studi hanno dimostrato che la specificità degli anticorpi PTM dell&#39;istone può essere altamente variabile e che questi reagenti presentano<html> Proprietà avorabili come il riconoscimento epitopico fuori bersaglio, forte influenza positiva e negativa da parte dei PTM vicini e difficoltà a discriminare l&#39;ordine di modifica su un determinato residuo ( ad esempio mono-, di- o tri-metililossina) 12 , 13 , 14 , 15 , 16 , 17 , 18 . Pertanto, è necessario un controllo rigoroso della qualità dei reagenti anticorpo specifico PTM-istone per interpretare con precisione i dati generati con questi reagenti preziosi. La tecnologia Microarray consente di interrogare simultaneamente migliaia di interazioni macromolecolari in un formato ad alta produttività, riproducibile e miniaturizzato. Per questo motivo, sono state create diverse piattaforme di microarray per analizzare il DNA proteico 19 ,&quot;&gt; 20, proteine-proteine 21 e proteine-peptide interazioni 22. In effetti, i microarray di peptide dell&#39;istone sono emerse come una piattaforma di scoperta informativa per la ricerca di biochimica cromatica, consentendo una profilazione ad alta velocità degli scrittori, cancellatori e lettori di istone PTMs 15 , 23 , 24 e anche per l&#39;analisi della specificità degli anticorpi degli istoni 17 , 25. Oltre alla loro applicazione nella ricerca di cromatina e epigenetica, gli array di peptone istonico hanno potenziale utilità come test diagnostico / prognostico per il lupus eritematoso sistemico e altre malattie autoimmuni, Gli anticorpi cromatici vengono generati 26 , 27 . Qui descriviamo una pipeline integrata che abbiamo sviluppato per la progettazione, la fabbricazione e la queMicroarrays di peptide istone per generare profili di specificità per gli anticorpi che riconoscono gli istoni e le loro PTM. Il gasdotto è facilitata da ArrayNinja, un open-source, un&#39;applicazione software interattivo che abbiamo recentemente sviluppato, che integra le fasi di progettazione e di analisi di esperimenti di microarray 28. ArrayNinja funziona meglio in Google Chrome. In breve, viene utilizzata una stampante microarray a contatto robotizzato per depositare una biblioteca di peptidi istoni coniugati biotina in posizioni definite su vetrini a microscopio vetrato con streptavidina. Le matrici possono quindi essere utilizzate in un modo conciso e in parallelo di test per interrogare le interazioni con gli anticorpi-epitopi ( Figura 1 ). La biblioteca del peptide è costituita da centinaia di peptidi sintetici unici che ospitano PTM (acetilazione lisina, metilazione lisina / arginina e fosforilazione serina / treonina) da soli e in combinazioni pertinenti in gran parte derivate da set di dati proteomici. Metodi per la sintesi e la convalida dei peptidi<html> Sono dettagliate altrove 23 . I dati generati dagli sforzi di screening degli anticorpi PTH istantanei in corso utilizzando questa piattaforma di array sono archiviati in una risorsa web pubblica, il database di specificità degli anticorpi Histone (www.histoneantibodies.com). In particolare, i microarray di peptide istone fabbricati con variazioni di questo protocollo sono stati ampiamente utilizzati per caratterizzare l&#39;attività dei domini di lettore di istone PTM 8 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 e più recentemente all&#39;istone del profilo PTM scrittore e attività gomma 24 . /files/ftp_upload/55912/55912fig1.jpg &quot;/&gt; Figura 1: Disposizione del fumetto della procedura stepwise per la screening degli anticorpi su un microarray di peptide dell&#39;istone. I peptidi biotinilati di istone che ospitano modifiche post-traslazionali definite (cerchi rossi e blu) sono stampati con biotina-fluoresceina sul vetro rivestito con streptavidina. Le interazioni positive sono visualizzate come fluorescenza rossa. <a href="http://ecsource.jove.com/files/ftp_upload/55912/55912fig1large.jpg" target="_blank">Clicca qui per visualizzare una versione più grande di questa figura.</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

Le modificazioni post-traduzionali (PTMs) sulle proteine ​​dell&#39;istone sono ampiamente studiate per i loro ruoli nella regolazione della struttura cromatica e dell&#39;espressione genica. La produzione di massa e la distribuzione di anticorpi specifici ai PTM istonici ha facilitato notevolmente la ricerca su questi segni. Poiché gli anticorpi PTM dell&#39;istone sono reagenti chiave per molte applicazioni di biochimica cromatica, è necessaria un&#39;analisi rigorosa della specificità degli anticorpi per un&#39;accurata interpretazione dei dati e un progresso continuo nel campo. Questo protocollo descrive una pipeline integrata per la progettazione, la fabbricazione e l&#39;uso di microarray peptidi per la profilazione della specificità degli anticorpi istoni. Gli aspetti di progettazione e analisi di questa procedura sono facilitati da ArrayNinja, un pacchetto software open source e interattivo che abbiamo recentemente sviluppato per semplificare la personalizzazione dei formati di stampa con microarray. Questa pipeline è stata utilizzata per la visualizzazione di un gran numero di anticorpi anti-PTM di istone commercialmente disponibili e ampiamente utilizzatiE i dati generati da questi esperimenti sono liberamente disponibili tramite un database di specificità anticorpo istante in linea e in espansione. Al di là degli istoni, la metodologia generale descritta qui può essere applicata ampiamente all&#39;analisi degli anticorpi PTM-specifici.

Questo protocollo è stato utilizzato per progettare e realizzare una piattaforma microarray peptide per l&#39;analisi della specificità degli anticorpi PTM. L&#39;array richiede una libreria di più di 300 funzionalità peptide uniche (20-40 residui di lunghezza) che rappresentano molte delle combinazioni note di PTM trovate sulle proteine ​​di istone del nucleo e delle varianti 38 . Questa pipeline è stata un cavallo di lavoro per lo screening di molti ant...

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

Analisi della specificità degli anticorpi istonici con microarray di peptide

Questo manoscritto descrive i metodi per applicare la tecnologia del microarray peptide alla profilazione di specificità degli anticorpi che riconoscono gli istoni e le loro modificazioni post-traslazionali.

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 Views.  Van Andel Research Institute.  Questo manoscritto descrive i metodi per applicare la tecnologia del microarray peptide alla profilazione di specificità degli anticorpi che riconoscono gli istoni e le loro modificazioni post-traslazionali. 

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.

Laboratorio scala di produzione e purificazione di un anticorpo terapeutico

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

Ricerca

Biochimica

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.

Facile manipolazione delle architetture in idrogel a base di proteine per applicazioni della coltura delle cellule

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.

Biopanning semi-automatizzato di librerie visualizzazione batterica per Peptide affinità reagente scoperta e l'analisi degli isolati risultanti

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.

La determinazione della specificità della proteasi nel Mouse tessuto estratti mediante spettrometria di massa MALDI-TOF: manipolazione di PH per causare i cambiamenti di specificità

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.

Screening dell'inibitore della kinasi in microarray proteici umane auto-assemblati

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.

Isolamento dell'istono dal tessuto fogliare di sorgo per la profilazione della spettrometria di massa dall'alto verso il basso dei potenziali marcatori epigenetici

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.

Rapida determinazione dell'affinità anticorpo-antigene mediante fotometria di massa

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.

Produzione cell-free di proteoliposomi per l'analisi funzionale e lo sviluppo di anticorpi mirati alle proteine di membrana

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.

Crio-EM e analisi a singola particella con Scipion

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.

In vitro Caratterizzazione degli Chaperoni degli istoni mediante saggi analitici, pull-down e chaperoning

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 ...