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1. Designing the Array Slide and Source Plate Layout
<ol>
	<li>Under the &#39;Plan a slide layout&#39; heading on the ArrayNinja interface, click on the link that corresponds to the microarray printer being used. 
	NOTE: ArrayNinja has been programmed to mimic the robotic movement of two commonly used microarray printers (see&#160;Table 1). Compatibility with the other arrayers can be configured upon request.</li>
	<li>Click inside the &#39;Load plate&#39; dialog box, type &#34;empty&#34;, and click &#39;enter.&#39; See&#160;Figure 1&#160;for a screenshot of the ArrayNinja design module.</li>
	<li>Adjust the spot diameter to 275 &#181;m and spot spacing to 375 &#181;m. Adjust the remaining settings (Plate Blocks/plate row, Total plate Rows, replicates, features in y, Super Arrays, SuperA fudge; see&#160;Figure 1) to customize how the features will appear on the microarray slide. 
	NOTE: The spot diameter is determined by the size of the microarray pin. As these settings are adjusted, the cartoon slide will update in real-time. Use this cartoon to preview how each setting is modifying the final slide layout.</li>
	<li>After the layout of features on the slide is finalized, mouse over each unique feature and enter the feature identifier in the pop-up dialog box. 
	NOTE: Feature identifiers can consist of numbers, letters, or combinations. This is only required for unique features, and a dialog box will not appear when replicates are selected.</li>
	<li>After all unique features have been assigned an identifier, click &#39;populate.&#39; Enter a name for the slide layout and click &#39;print your plate&#39; to save. A new page will open displaying the number of 384-well source plates needed to fabricate the chosen slide design and a table mapping the physical location of each feature to be loaded in the source plate(s). 
	NOTE: Remember this name, as it will be used to recall this design when analyzing microarray data. Clicking &#39;print your plate&#39; saves the layout within ArrayNinja.</li>
</ol>
2. Fabricating Microarrays
<ol>
	<li>Preparing the Source Plate
	<ol>
		<li>Use the map generated with ArrayNinja to create the 384-well source plate(s). 
		&#8203;NOTE: Detailed descriptions of peptides queried on this platform can be found elsewhere.</li>
		<li>Deposit 1 - 2 &#181;L of each feature (e.g., biotinylated histone peptide) into the correct well of the 384-well source plate(s). 
		NOTE: Biotinylated histone peptides are typically deposited from 200 - 400 &#181;M stock solutions, which equates to 10 to 25-fold molar excess of the peptide to streptavidin binding sites in a single array spot. This is calculated using the equation: 
		<img alt="Equation 1" src="/files/ftp_upload/22115/22115_eq01.jpg" style="margin: auto;" /> 
		where V is the volume delivered by a pin, [P] is the concentration of the feature being printed, NA is Avogadro&#39;s number, and S is the surface area of a spot. C is the coverage of the slide, expressed as the number of streptavidin molecules per unit area multiplied by three (the average number of available streptavidin binding sites). V and C are obtained by the respective manufacturer&#39;s. Other features may require different concentrations depending on the size of the biomolecule where crowding may be a concern. A range of printing concentrations for each new type of feature should be tested empirically to determine the optimum printing concentration.</li>
		<li>Dilute each feature 10-fold with 1x printing buffer supplemented with 1% bovine serum albumin (BSA). Spin the source plate(s) at 500 x g for 2 min at room temperature. 
		NOTE: The inclusion of fluorescein-labeled biotin (5 &#181;g/&#181;L) in the printing buffer is recommended as a spotting control and as a visual aid to facilitate proper array alignment during analysis (see Figure 3).</li>
	</ol>
	</li>
	<li>Printing Protocol (Figure 2A &#8211; B).
	<ol>
		<li>Prepare the arrayer by emptying the waste collection container and filling the wash solution container and humidifier container with sterile distilled water.</li>
		<li>Enter the parameters used to design the slide in ArrayNinja (Figure 1) into the microarray printer control program.</li>
		<li>Using the microarray printer control program, set the washing procedure for a 1 s wash with one immersion. Set the post-wash settings to re-dip the pins 5 times following each wash. Set the humidity to 60%. 
		NOTE: The optimal wash configuration may vary depending on the microarray printer being used. The optimal post-wash pin dip configuration may vary depending on the microarray printer being used.</li>
		<li>Insert functionalized slides (e.g., streptavidin-coated glass) into the substrate platens and place all platens into the platen elevator. Insert the source plate(s) into the plate holder(s) and place into the source plate elevator.</li>
		<li>Click &#39;print&#39;. Monitor the printing process for a few rounds of feature deposition to ensure all wash and dip settings are correct. When the print run is complete, remove the substrate platens from the arrayer. 
		NOTE: When large print runs prohibit completion of blocking steps within one day, printed slides can be incubated in a humidified chamber at 4 &#176;C overnight. Incubate the slides next to a small beaker of water inside a cardboard box sealed with plastic wrap.</li>
		<li>Block the slides with blocking buffer for 30 min at room temperature with mixing.</li>
		<li>Wash the slides 2 x 10 min at room temperature in phosphate buffered saline (PBS), pH 7.6 with mixing. Dry the slides by spinning in a microarray slide centrifuge for 30 s at room temperature. 
		NOTE: For processing a large number of slides at once, a high-throughput microscope slide washing chamber can be used, allowing 50 slides to be washed in parallel.</li>
		<li>For slides designed to be partitioned with wax, proceed to section 2.1. For all other designs, store slides at 4 &#176;C protected from light and moisture. 
		NOTE: Printed biotinylated histone peptides are stable for at least 6 months when stored this way.</li>
	</ol>
	</li>
</ol>
2. Partitioning Microarray Slides
<ol>
	<li>Hydrophobic Wax Pen (Figure 2C)
	<ol>
		<li>Apply wax around the areas that contain features using a wax pen. Allow wax to air dry for 5 min before proceeding to section 3. 
		NOTE: After blocking, the array spots can be very difficult to visualize by eye. The slide design from ArrayNinja can be printed to scale and used as a guide for applying wax.</li>
	</ol>
	</li>
	<li>Silicon gasket (Figure 2D)
	<ol>
		<li>Peel the clear film off the back of the array gasket and place adhesive side down onto the microarray slide.</li>
		<li>Hold the gasket in place for 5 s prior to proceeding to section 3.</li>
	</ol>
	</li>
	<li>Wax Imprint (Figure 2E)
	<ol>
		<li>For slides designed to be partitioned by wax, print a test slide on plain glass using 10% BSA. Use this test slide to optimize the wax imprinter guides or array settings to ensure all features will be within the wax-mold chambers.</li>
		<li>Heat the microarray wax imprinter to 85 &#176;C until all the wax is completely melted, approximately 30 min.</li>
		<li>Insert the slide with the printed side facing down and push the slide all the way to the right guide on the microarray imprinter. Pull up the lever to bring the mold into contact with the surface of the slide. Hold for 2 s. 
		NOTE: The hold time can be altered to achieve optimal wax border thickness.</li>
		<li>Quickly remove the slide and visually inspect the wax borders to ensure all wells are enclosed and that the borders are not so thick that they encroach on the spots. Store slides at 4 &#176;C protected from moisture and light. 
		NOTE: The hold time can be increased or decreased to obtain thicker or thinner borders.</li>
	</ol>
	</li>
</ol>
3. Hybridizing a Histone PTM Antibody with a Peptide Microarray
<ol>
	<li>Prepare hybridization buffer (PBS, pH 7.6, 5% BSA, 0.1% Tween-20).</li>
	<li>Equilibrate the slide in hybridization buffer using a hybridization vessel. Completely cover the entire slide in hybridization buffer and incubate for 30 min at 4 &#176;C on an orbital shaker at low speed.</li>
	<li>Prepare a solution containing diluted histone PTM antibody in hybridization buffer. 
	NOTE: In the example data, both histone Antibody #1 and Antibody #2 were diluted 1:1,000 in hybridization buffer. A dilution range similar to that used for immunoblotting is recommended as a starting point.</li>
	<li>Incubate the array with the antibody solution for 1 h at 4 &#176;C. Remove the antibody solution and wash the array 3 times for 5 min at 4 &#176;C with cold PBS, pH 7.6.</li>
	<li>Prepare a 1:5,000 - 1:10,000 dilution of fluorescent dye-conjugated secondary antibody in hybridization buffer.</li>
	<li>Incubate the array with secondary antibody solution for 30 min at 4 &#176;C protected from light. Remove the secondary antibody solution and wash the microarray slide 3 times for 5 min at 4 &#176;C with PBS, pH 7.6. Dip the microarray in a 50-mL conical tube containing 0.1x PBS, pH 7.6 to remove excess salt at room temperature. Dry the slide in a microarray slide centrifuge at room temperature.</li>
	<li>Scan the slide with a microarray scanner at 25 &#181;m resolution or higher, following the microarray scanner manufacturer&#39;s recommended scanning protocol. 
	NOTE: If fluorescein-labeled biotin tracer is present, scan both the green channel (ex: 488 nm, em: 509 nm) and the channel that corresponds to the fluorescent dye-conjugated secondary antibody, typically red (ex: 635 nm, em: 677 nm). The goal of scanning is to obtain single channel .tif files that can be merged into a single .png file.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Printing Buffer</td>
			<td>ArrayIt</td>
			<td>PPB</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Omnipure</td>
			<td>2390</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidin-coated glass microscope slides</td>
			<td>Greiner Bio-one</td>
			<td>439003-25</td>
			<td></td>
		</tr>
		<tr>
			<td>polypropylene 384 well plate</td>
			<td>Greiner Bio-one</td>
			<td>784201</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotin-fluorescein</td>
			<td>Sigma</td>
			<td>53608</td>
			<td></td>
		</tr>
		<tr>
			<td>contact microarray printer</td>
			<td>Aushon</td>
			<td>2470</td>
			<td>Aushon 2470 Microarray Printer</td>
		</tr>
		<tr>
			<td>contact microarray printer</td>
			<td>Gene Machines</td>
			<td>OmniGrid 100</td>
			<td>OmniGrid Microarray Printer</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Invitrogen</td>
			<td>14190</td>
			<td></td>
		</tr>
		<tr>
			<td>Blocking Buffer</td>
			<td>ArrayIt</td>
			<td>SBB</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrophobic wax pen</td>
			<td>Vector Labs</td>
			<td>H-4000</td>
			<td>ImmEdge Hydrophobic Barrier PAP Pen</td>
		</tr>
		<tr>
			<td>Silicon Gasket</td>
			<td>Grace Bio-labs</td>
			<td>622511</td>
			<td></td>
		</tr>
		<tr>
			<td>Hybridization Vessel</td>
			<td>Thermo Scientific</td>
			<td>267061</td>
			<td>or similar vessel</td>
		</tr>
		<tr>
			<td>Fluorescent-dye conjugated secondary antibody</td>
			<td>Life Technologies</td>
			<td>A-21244</td>
			<td>Alexa Fluor 647 (anti-rabbit)</td>
		</tr>
		<tr>
			<td>Fluorescent-dye conjugated secondary antibody</td>
			<td>Life Technologies</td>
			<td>A-21235</td>
			<td>Alexa Fluor 647 (anti-mouse)</td>
		</tr>
		<tr>
			<td>Wax Imprinter</td>
			<td>ArrayIt</td>
			<td>MSI48</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Omnipure</td>
			<td>9490</td>
			<td></td>
		</tr>
		<tr>
			<td>Microarray Scanner</td>
			<td>Innopsys</td>
			<td>InnoScan 1100AL</td>
			<td>or equivalent microarray scanner</td>
		</tr>
		<tr>
			<td>EipTitan Histone Peptide Microarray</td>
			<td>Epicypher</td>
			<td>112001</td>
			<td></td>
		</tr>
		<tr>
			<td>AbSurance Pro Histone Peptide Microarray</td>
			<td>Millipore</td>
			<td>16668</td>
			<td></td>
		</tr>
		<tr>
			<td>MODified Histone Peptide Array</td>
			<td>Active Motif</td>
			<td>13001</td>
			<td></td>
		</tr>
		<tr>
			<td>Histone Code Peptide Microarrays</td>
			<td>JPT</td>
			<td>His_MA_01</td>
			<td></td>
		</tr>
		<tr>
			<td>Wax</td>
			<td>Royal Oak</td>
			<td>GulfWax</td>
			<td>for wax imprinter</td>
		</tr>
		<tr>
			<td>Humidified Microarray Slide Hybridization Chamber</td>
			<td>VWR</td>
			<td>97000-284</td>
			<td></td>
		</tr>
		<tr>
			<td>High throughput microscope slide washing chamber</td>
			<td>ArrayIt</td>
			<td>HTW</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope slide centrifuge</td>
			<td>VWR</td>
			<td>93000-204</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody 1</td>
			<td>Abcam</td>
			<td>8898</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody 2</td>
			<td>Millipore</td>
			<td>07-473</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotinylated histone peptide</td>
			<td>EpiCypher</td>
			<td>12-2001</td>
			<td>Example peptide. Similar peptides with various modifications are available from several commercial sources.</td>
		</tr>
		<tr>
			<td>ImageMagick</td>
			<td></td>
			<td></td>
			<td>https://www.imagemagick.org/script/index.php</td>
		</tr>
		<tr>
			<td>ArrayNinja</td>
			<td></td>
			<td></td>
			<td>https://rothbartlab.vai.org/tools/</td>
		</tr>
	</tbody>
</table>

a peptide microarray technology for specificity profiling of antibodies

Source: Cornett, E. M., et al. <a href="https://app.jove.com/v/55912">Analysis of Histone Antibody Specificity with Peptide Microarrays.</a> J. Vis. Exp. (2017).
This video demonstrates a technique that applies peptide microarray technology to profile the specificity of antibodies recognizing post-translational modifications (PTMs) on histone peptides. A microarray slide containing immobilized fluorophore-conjugated histone peptides with known PTM combinations is treated with target PTM-specific antibodies. This process helps identify the antibody&#39;s specificity for the target and its potential cross-reactivity with non-target PTMs.

<img alt="Figure 1" src="/files/ftp_upload/22115/22115_Figure1.jpg" /> 
Figure 1: ArrayNinja Design Module. A screen shot of the ArrayNinja design module is shown in the dotted line. The control panel (top) shows all of the parameters that can be altered on the microarray printer. As these parameters are adjusted, the cartoon image of the slide layout (bottom left) updates in real time. After the layout is set, the user can mouse over individual spots to enter unique...

A Peptide Microarray Technology for Specificity Profiling of Antibodies

1. Designing the Array Slide and Source Plate Layout

	Under the &#39;Plan a slide layout&#39; heading on the ArrayNinja interface, click on the link that ...

Take a microarray slide containing histone peptides with target and non-target post-translational modifications or PTMs.
The peptides are immobilized via conjugated biotin at specific spots on the streptavidin-functionalized glass slide.
The spots also contain an immobilized biotin-conjugated green fluorophore, facilitating the identification of the spots.
Introduce a blocking buffer, preventing non-specific antibody interaction.
Remove the buffer and centrifuge to dry the slide.
Inside a wax imprinter, border the array with wax.
Equilibrate the array with a hybridization buffer.
Incubate with the primary antibodies specific for a target PTM.
Introduce red fluorophore-conjugated secondary antibodies that bind to the primary antibodies.
Remove unbound antibodies and centrifuge to dry the slide.
Image the slide using a microarray scanner. The green fluorescence helps identify the spots, while the red fluorescence indicates PTM-antibody interaction, demonstrating antibody specificity.
Recognition of the target PTM demonstrates antibody specificity, whereas recognition of non-target PTMs indicates antibody cross-reactivity.

a-peptide-microarray-technology-for-specificity-profiling

Research

JoVE Encyclopedia of Experiments

Immunology

Antibody-Based Technologies

Characterization and Functional Analysis

44 vistas. Fuente: Cornett, E. M., et al. Análisis de la especificidad de anticuerpos de histonas con microarrays de péptidos. J. Vis. Exp. (2017). Este video muestra una técnica que aplica la tecnología de microarrays de péptidos para perfilar la especificidad de los anticuerpos reconociendo modificaciones postraduccionales (PTM) en péptidos de histonas. Un portaobjetos de microarray que contiene péptidos de histonas conjugados con fluoróforos inmovilizados con combinaciones conocidas de PTM se t...

Video: Una tecnología de microarrays de péptidos para el perfil de especificidad de los anticuerpos - Concepto

Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform

Early embryonic loss is a large contributor to infertility in cattle. Moreover, bovine becomes an interesting model to study human preimplantation embryo development due to their similar developmental process. Although genetic factors are known to affect early embryonic development, the discovery of such factors has been a serious challenge. Microarray technology allows quantitative measurement and gene expression profiling of transcript levels on a genome-wide basis. One of the main decisions that have to be made when planning a microarray experiment is whether to use a one- or two-color approach. Two-color design increases technical replication, minimizes variability, improves sensitivity and accuracy as well as allows having loop designs, defining the common reference samples. Although microarray is a powerful biological tool, there are potential pitfalls that can attenuate its power. Hence, in this technical paper we demonstrate an optimized protocol for RNA extraction, amplification, labeling, hybridization of the labeled amplified RNA to the array, array scanning and data analysis using the two-color analysis strategy.

Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform

Microarray technology allows quantitative measurement and gene expression profiling of transcripts on a genome-wide basis. Therefore, this protocol provides an optimized technical procedure in a two-color custom made bovine array using Day 7 bovine embryos to demonstrate the feasibility of using low amount of total RNA.

Transcriptoma de perfiles de<em&gt; In-Vivo</em&gt; Productos fabricados preimplantación de embriones bovinos mediante plataforma de microarrays de dos colores

Early embryonic loss is a large contributor to infertility in cattle. Moreover, bovine becomes an interesting model to study human preimplantation embryo ...

Investigación

JoVE Journal

Biología del Desarrollo

Extracellular Protein Microarray Technology for High Throughput Detection of Low Affinity Receptor-Ligand Interactions

Secreted factors, membrane-tethered receptors, and their interacting partners are main regulators of cellular communication and initiation of signaling cascades during homeostasis and disease, and as such represent prime therapeutic targets. Despite their relevance, these interaction networks remain significantly underrepresented in current databases; therefore, most extracellular proteins have no documented binding partner. This discrepancy is primarily due to the challenges associated with the study of the extracellular proteins, including expression of functional proteins, and the weak, low affinity, protein interactions often established between cell surface receptors. The purpose of this method is to describe the printing of a library of extracellular proteins in a microarray format for screening of protein-protein interactions. To enable detection of weak interactions, a method based on multimerization of the query protein under study is described. Coupled to this microbead-based multimerization approach for increased multivalency, the protein microarray allows robust detection of transient protein-protein interactions in high throughput. This method offers a rapid and low sample consuming-approach for identification of new interactions applicable to any extracellular protein. Protein microarray printing and screening protocol are described. This technology will be useful for investigators seeking a robust method for discovery of protein interactions in the extracellular space.

Here, we present a protocol to screen extracellular protein microarrays for identification of novel receptor-ligand interactions in high throughput. We also describe a method to enhance detection of transient protein-protein interactions by using protein-microbead complexes.

Tecnología de microarrays de proteínas extracelulares para detección de alto rendimiento de las interacciones ligando-Receptor de baja afinidad

Secreted factors, membrane-tethered receptors, and their interacting partners are main regulators of cellular communication and initiation of signaling ...

Bioquímica

Basophil Activation Test for Allergy Diagnosis

The basophil activation test (BAT) is a complementary in vitro diagnostic test that can be used in addition to clinical history, skin test (ST), and specific IgE (sIgE) determination in the evaluation of IgE-mediated allergic reactions to food, insect venom, drugs, as well as some forms of chronic urticaria. However, the role of this technique in the diagnostic algorithms is highly variable and not well determined.
BAT is based on the determination of basophil response to allergen/drug cross-linking IgE activation through the measurement of activation markers (such as CD63, CD203c) by flow cytometry. This test can be a useful and complementary tool to avoid controlled challenge tests&#160;to confirm allergy diagnosis, especially in subjects experiencing severe life-threatening reactions. In general, the performance of BAT should be considered if i) the allergen/drug produces false positive results in ST; ii) there is no allergen/drug source to use for ST or sIgE determination; iii) there is discordance between patient history and ST or sIgE determination; iv) symptoms suggest that ST may result in systemic response; v) before considering a CCT to confirm the culprit allergen/drug. The main limitations of the test are related to non-optimal sensitivity, especially in drug allergy, the need to perform the test no longer than 24 h after sample extraction, and the lack of standardization between laboratories in terms of procedures, concentrations, and cell markers.

The basophil activation test is a complementary in vitro diagnostic test for the evaluation of IgE-mediated allergic reactions based on the detection of basophil activation in the presence of a specific stimulus through the measure of activation markers by flow cytometry.

Prueba de activación de basófilos para el diagnóstico de alergia

The basophil activation test (BAT) is a complementary in vitro diagnostic test that can be used in addition to clinical history, skin test (ST), and ...

A Peptide Microarray Technology for Specificity Profiling of Antibodies

Transcripción

A Peptide Microarray Technology for Specificity Profiling of Antibodies