Name: chemically-blocked antibody microarray for multiplexed high-throughput profiling of specific protein glycosylation in complex samples
Uploaded: 2012-05-04T13:00:00
Description: Watch this Scientific Journal Video about Chemically-blocked Antibody Microarray for Multiplexed High-throughput Profiling of Specific Protein Glycosylation in Complex Samples at JoVE.com

This work was supported by the Institute for Hepatitis and Virus Research.

1. Print an Antibody Microarray for the Assay
<ol>
	<li>Dilute all antibodies to 0.5 mg/ml in phosphate buffer saline, ph 7.2 (PBS).</li>
	<li>Aliquot 40 &mu;l of each antibody into the 384-well source plate.</li>
	<li>Load the 384-well source plate onto the Scienion sciFLEXARRAYER microarrayer.</li>
	<li>Load 20 PATH microarray slides onto the microarrayer as target.</li>
	<li>Set the microarrayer to print 48 identical subarrays, in which 27 antibodies and control proteins are spotted in triplicate in a 9x9 pattern (<...

<ol>
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K., Wingren, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+proteomics+using+antibody+microarrays:+an+update.">High-throughput proteomics using antibody microarrays: an update.</a> Expert Review of Molecular Diagnostics. 7, 673-686 (2007).</li><li>Sanchez-Carbayo, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibody+array-based+technologies+for+cancer+protein+profiling+and+functional+proteomic+analyses+using+serum+and+tissue+specimens.">Antibody array-based technologies for cancer protein profiling and functional proteomic analyses using serum and tissue specimens.</a> Tumor Biology. 31, 103-112 (2010).</li><li>Porter, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+motif-based+analysis+of+glycan+array+data+to+determine+the+specificities+of+glycan-binding+proteins.">A motif-based analysis of glycan array data to determine the specificities of glycan-binding proteins.</a> Glycobiology. 20, 369-380 (2010).</li><li>Maupin, K. 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1. Target protein and capture antibody selection
Prior to the antibody microarray assay, some reagents and materials are needed to be considered and prepared. To design an antibody microarray for glycan profiling or glycan biomarker screening, a panel of antibodies specific to glycoprotein candidates should be determined according to the literature or from previous results. These antibodies were usually purchased from different vendors such as R&amp;D Systems etc. IgGs are preferred capture antib...

<table border="1">
	<tbody>
		<tr>
			<td>ID</td>
			<td>Name of the reagent</td>
			<td>Abbreviation</td>
			<td>Company</td>
			<td>Catalog #</td>
		</tr>
		<tr>
			<td>L1</td>
			<td>Biotinylated Concanavalin A</td>
			<td>ConA</td>
			<td>Vector Laboratories</td>
			<td>BK-1000</td>
		</tr>
		<tr>
			<td>L2</td>
			<td>Biotinylated Sambucus Nigra Lectin</td>
			<td>SNA</td>
			<td>Vector Laboratories</td>
			<td>B-1305</td>
		</tr>
		<tr>
			<td>L3</td>
			<td>Biotinylated Lens Culinaris Agglutinin</td>
			<td>LCA</td>
			<td>Vector Laboratories</td>
			<td>BK-2000</td>
		</tr>
		<tr>
			<td>L4</td>
			<td>Biotinylated Ricinus Communis Agglutinin I</td>
			<td>RCA</td>
			<td>Vector Laboratories</td>
			<td>BK-1000</td>
		</tr>
		<tr>
			<td>L5</td>
			<td>Biotinylated Aleuria Aurantia Lectin</td>
			<td>AAL</td>
			<td>Vector Laboratories</td>
			<td>B-1395</td>
		</tr>
		<tr>
			<td>L6</td>
			<td>Biotinylated Erythrina Cristagalli Lectin</td>
			<td>ECL</td>
			<td>Vector Laboratories</td>
			<td>BK-3000</td>
		</tr>
		<tr>
			<td>L7</td>
			<td>Biotinylated Griffonia (Bandeiraea) Simplicifolia Lectin II</td>
			<td>GSL II</td>
			<td>Vector Laboratories</td>
			<td>BK-3000</td>
		</tr>
		<tr>
			<td>L8</td>
			<td>Biotinylated Wheat Germ Agglutinin</td>
			<td>WGA</td>
			<td>Vector Laboratories</td>
			<td>BK-1000</td>
		</tr>
		<tr>
			<td>L9</td>
			<td>Biotinylated Phaseolus vulgaris Erythroagglutinin</td>
			<td>PHA-E</td>
			<td>Vector Laboratories</td>
			<td>BK-2000</td>
		</tr>
		<tr>
			<td>L10</td>
			<td>Biotinylated Phaseolus vulgaris Leucoagglutinin</td>
			<td>PHA-L</td>
			<td>Vector Laboratories</td>
			<td>BK-2000</td>
		</tr>
		<tr>
			<td>L11</td>
			<td>Biotinylated Peanut Agglutinin</td>
			<td>PNA</td>
			<td>Vector Laboratories</td>
			<td>BK-1000</td>
		</tr>
		<tr>
			<td>L12</td>
			<td>Biotinylated Pisum Sativum Agglutinin</td>
			<td>PSA</td>
			<td>Vector Laboratories</td>
			<td>BK-2000</td>
		</tr>
		<tr>
			<td>L13</td>
			<td>Biotinylated Dolichos Biflorus Agglutinin</td>
			<td>DBA</td>
			<td>Vector Laboratories</td>
			<td>BK-1000</td>
		</tr>
		<tr>
			<td>L14</td>
			<td>Biotinylated Datura Stramonium Lectin</td>
			<td>DSL</td>
			<td>Vector Laboratories</td>
			<td>BK-3000</td>
		</tr>
		<tr>
			<td>L15</td>
			<td>Biotinylated Sophora Japonica Agglutinin</td>
			<td>SJA</td>
			<td>Vector Laboratories</td>
			<td>BK-2000</td>
		</tr>
		<tr>
			<td>L16</td>
			<td>Biotinylated Soybean Agglutinin</td>
			<td>SBA</td>
			<td>Vector Laboratories</td>
			<td>BK-1000</td>
		</tr>
		<tr>
			<td>L17</td>
			<td>Biotinylated Solanum Tuberosum (Potato) Lectin</td>
			<td>STL</td>
			<td>Vector Laboratories</td>
			<td>BK-3000</td>
		</tr>
		<tr>
			<td>L18</td>
			<td>Biotinylated Griffonia (Bandeiraea) Simplicifolia Lectin I</td>
			<td>GSL I</td>
			<td>Vector Laboratories</td>
			<td>BK-2000</td>
		</tr>
		<tr>
			<td>L19</td>
			<td>Biotinylated Vicia Villosa Lectin</td>
			<td>VVL</td>
			<td>Vector Laboratories</td>
			<td>BK-2000</td>
		</tr>
		<tr>
			<td>L20</td>
			<td>Biotinylated Lycopersicon Esculentum (Tomato) Lectin</td>
			<td>LEL</td>
			<td>Vector Laboratories</td>
			<td>BK-3000</td>
		</tr>
		<tr>
			<td>L21</td>
			<td>Biotinylated Ulex Europaeus Agglutinin I</td>
			<td>UEA I</td>
			<td>Vector Laboratories</td>
			<td>BK-1000</td>
		</tr>
		<tr>
			<td>L22</td>
			<td>Biotinylated Jacalin</td>
			<td>JACALIN</td>
			<td>Vector Laboratories</td>
			<td>BK-3000</td>
		</tr>
		<tr>
			<td>A1</td>
			<td>Goat F(ab')2 Fragment anti-human IgM, Fc5&mu; antibody</td>
			<td>IgM</td>
			<td>Jackson Immuno Research</td>
			<td>109-006-129</td>
		</tr>
		<tr>
			<td>A2</td>
			<td>Donkey F(ab')2 Frag anti-human IgG (H+L) antibody</td>
			<td>AB1</td>
			<td>Jackson Immuno Research</td>
			<td>709-006-149</td>
		</tr>
		<tr>
			<td>A3</td>
			<td>Mouse anti-human IgG F(ab')2 monoclonal antibody</td>
			<td>AB3</td>
			<td>Jackson Immuno Research</td>
			<td>209-005-097</td>
		</tr>
		<tr>
			<td>A4</td>
			<td>Goat anti-human alpha 2 macroglobulin polyclonal antibody</td>
			<td>A2M</td>
			<td>GeneTex</td>
			<td>GTX62924</td>
		</tr>
		<tr>
			<td>A5</td>
			<td>Rabbit anti-human alpha-1-antitrypsin polyclonal antibody</td>
			<td>A1AT</td>
			<td>Lee Biosiences</td>
			<td>CA1T-80A</td>
		</tr>
		<tr>
			<td>A6</td>
			<td>Mouse anti-human alpha-1-antitrypsin monoclonal antibody</td>
			<td>A1AT</td>
			<td>Sigma Aldrich</td>
			<td>SAB4200198</td>
		</tr>
		<tr>
			<td>A7</td>
			<td>Rabbit anti-human alpha-1-antitrypsin polyclonal antibody</td>
			<td>ACT</td>
			<td>NeoMarkers</td>
			<td>RB-367-A1</td>
		</tr>
		<tr>
			<td>A8</td>
			<td>Rabbit anti-human alpha-1-antichymotrypsin polyclonal antibody</td>
			<td>ACT</td>
			<td>Fisher Scientific</td>
			<td>RB9213R7</td>
		</tr>
		<tr>
			<td>A9</td>
			<td>Mouse anti-human transferrin monoclonal antibody</td>
			<td>Transferrin</td>
			<td>GeneTex</td>
			<td>GTX101035</td>
		</tr>
		<tr>
			<td>A10</td>
			<td>Rabbit anti-human transferrin polyclonal antibody</td>
			<td>Transferrin</td>
			<td>GeneTex</td>
			<td>GTX77130</td>
		</tr>
		<tr>
			<td>A11</td>
			<td>Goat anti-human apolipoprotein J polyclonal antibody</td>
			<td>ApoJ</td>
			<td>Abcam</td>
			<td>ab7610</td>
		</tr>
		<tr>
			<td>A12</td>
			<td>Mouse anti-human GP73 monoclonal antibody</td>
			<td>GP73</td>
			<td>Abbott</td>
			<td>14H4-23</td>
		</tr>
		<tr>
			<td>A13</td>
			<td>Mouse anti-human GP73 monoclonal antibody</td>
			<td>GP73</td>
			<td>SANTA CRUZ BIOTECHNOLOGY INC</td>
			<td>sc-101275</td>
		</tr>
		<tr>
			<td>A14</td>
			<td>Rabbit anti-human alpha-1 fetoprotein polyclonal antibody</td>
			<td>AFP</td>
			<td>GenWay</td>
			<td>GWB-41C966</td>
		</tr>
		<tr>
			<td>A15</td>
			<td>Mouse anti-human alpha-1 fetoprotein monoclonal antibody</td>
			<td>AFP</td>
			<td>Fitzgerald</td>
			<td>10-A05A</td>
		</tr>
		<tr>
			<td>A16</td>
			<td>Mouse anti-human hemopexin monoclonal antibody</td>
			<td>Hemopexin</td>
			<td>Assaypro</td>
			<td>60190-05011</td>
		</tr>
		<tr>
			<td>A17</td>
			<td>Mouse anti-human glypican-3(1G12) monoclonal antibody</td>
			<td>GPL3</td>
			<td>Santa Cruz Bio</td>
			<td>sc-65443</td>
		</tr>
		<tr>
			<td>A18</td>
			<td>Mouse anti-human Kininogen (LMW) monoclonal antibody</td>
			<td>Kininogen</td>
			<td>Assaypro</td>
			<td>20333-05011</td>
		</tr>
		<tr>
			<td>A19</td>
			<td>Rabbit anti-human MMP-21 monoclonal antibody</td>
			<td>MMP21</td>
			<td>Epitomic</td>
			<td>1955-1</td>
		</tr>
		<tr>
			<td>A20</td>
			<td>Mouse anti-human CEACAM-1 monoclonal antibody</td>
			<td>CEACAM</td>
			<td>R&amp;D Systems</td>
			<td>MAB1180</td>
		</tr>
		<tr>
			<td>A21</td>
			<td>Rat anti-human DPPIV/CD26 monoclonal antibody</td>
			<td>DPPIV</td>
			<td>R&amp;D Systems</td>
			<td>MAB22441</td>
		</tr>
		<tr>
			<td>A22</td>
			<td>Mouse anti-human PIVKA II monoclonal antibody</td>
			<td>PIVICA</td>
			<td>Crystal chem</td>
			<td>8040</td>
		</tr>
		<tr>
			<td>A23</td>
			<td>Mouse anti-carcinoembryonic antigen</td>
			<td>CEA</td>
			<td>US biological</td>
			<td>C1300</td>
		</tr>
		<tr>
			<td>A24</td>
			<td>Mouse anti-CA125 Cancer Antigen</td>
			<td>CA125</td>
			<td>US biological</td>
			<td>C0050-01D</td>
		</tr>
		<tr>
			<td>A25</td>
			<td>Mouse anti -CA19-9 Cancer antigen</td>
			<td>CA19-9</td>
			<td>US biological</td>
			<td>C0075-18</td>
		</tr>
		<tr>
			<td>A26</td>
			<td>Mouse anti-Lewis x monoclonal antibody</td>
			<td>Lewis X</td>
			<td>Calbiochem</td>
			<td>434631</td>
		</tr>
		<tr>
			<td>bio</td>
			<td>Biotinylated BSA (positive control)</td>
			<td>Bio</td>
			<td>Home-made</td>
			<td>N/A</td>
		</tr>
	</tbody>
</table>
Table 1. List of lectins and antibodies used in this protocol.
<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent s/equipments</td>
			<td>Company</td>
			<td>Catalogue number</td>
		</tr>
		<tr>
			<td>Non contact microarrayer</td>
			<td>BioDot Inc</td>
			<td>sciFLEXARRAYER</td>
		</tr>
		<tr>
			<td>384 microplate</td>
			<td>Fisher</td>
			<td>14-230-243</td>
		</tr>
		<tr>
			<td>FoodSaver</td>
			<td>FoodSaver</td>
			<td>V3835</td>
		</tr>
		<tr>
			<td>Ultrathin nitrocellulose coate microarray slides</td>
			<td>Gentel</td>
			<td>PATH</td>
		</tr>
		<tr>
			<td>Slide Imprinter (optional)</td>
			<td>The Gel Company</td>
			<td>WSP60-1</td>
		</tr>
		<tr>
			<td>Shaker</td>
			<td>Fisher</td>
			<td>15-453-211</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5804 000.013</td>
		</tr>
		<tr>
			<td>Slide washing basin/Slide Staining Dish with Removable Rack</td>
			<td>Fisher</td>
			<td>08-812</td>
		</tr>
		<tr>
			<td>Slide incubation chamber/microscope slide box</td>
			<td>Fisher</td>
			<td>03-448-5</td>
		</tr>
		<tr>
			<td>Brij 35, 30 w/v% solution in water</td>
			<td>Acros Organics</td>
			<td>AC32958-0025</td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Fisher</td>
			<td>P337-100</td>
		</tr>
		<tr>
			<td>Sodium Periodate (NaIO4)</td>
			<td>Sigma</td>
			<td>311448</td>
		</tr>
		<tr>
			<td>L-Glutamic acid &gamma;-hydrazide</td>
			<td>Sigma</td>
			<td>G-7257</td>
		</tr>
		<tr>
			<td>Sodium Acetate Anhydrous (CH3COONa)</td>
			<td>Sigma</td>
			<td>S2889</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Lampire Biological Labs</td>
			<td>7500804</td>
		</tr>
		<tr>
			<td>Phosphate Buffer Saline (PBS) (10X)</td>
			<td>Denville Scientific</td>
			<td>CP4390-48</td>
		</tr>
		<tr>
			<td>Dylight 549 conjugated NeutrAvidin</td>
			<td>Thermo</td>
			<td>22837</td>
		</tr>
		<tr>
			<td>Protease Inhibitor Cocktail Tablets</td>
			<td>Roche</td>
			<td>4693159001</td>
		</tr>
		<tr>
			<td>ChromPure Human IgG, Fc fragment</td>
			<td>Jackson Immunoresearch</td>
			<td>009-000-008</td>
		</tr>
		<tr>
			<td>ChromPure Human IgG, whole molecule</td>
			<td>Jackson Immunoresearch</td>
			<td>009-000-003</td>
		</tr>
		<tr>
			<td>ChromPure Mouse IgG, whole molecule</td>
			<td>Jackson Immunoresearch</td>
			<td>015-000-003</td>
		</tr>
		<tr>
			<td>ChromPure Mouse IgG, Fc fragment</td>
			<td>Jackson Immunoresearch</td>
			<td>015-000-008</td>
		</tr>
		<tr>
			<td>ChromPure Rabbit IgG, whole molecule</td>
			<td>Jackson Immunoresearch</td>
			<td>011-000-003</td>
		</tr>
		<tr>
			<td>ChromPure Donkey IgG, whole molecule</td>
			<td>Jackson Immunoresearch</td>
			<td>017-000-003</td>
		</tr>
		<tr>
			<td>Microarray Scanner</td>
			<td>Tecan</td>
			<td>LS Reloaded</td>
		</tr>
	</tbody>
</table>
Table 2. List of equipments and reagents used in this protocol.

chemically-blocked antibody microarray for multiplexed high-throughput profiling of specific protein glycosylation in complex samples

In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that allows for the glycosylation profiling of specific proteins. Glycosylation of proteins is the most prevalent post-translational modification found on proteins, and leads diversified modifications of the physical, chemical, and biological properties of proteins. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases. However, current methods to study protein glycosylation typically are too complicated or expensive for use in most normal laboratory or clinical settings and a more practical method to study protein glycosylation is needed. The new protocol described in this study makes use of a chemically blocked antibody microarray with glycan-binding protein (GBP) detection and significantly reduces the time, cost, and lab equipment requirements needed to study protein glycosylation. In this method, multiple immobilized glycoprotein-specific antibodies are printed directly onto the microarray slides and the N-glycans on the antibodies are blocked. The blocked, immobilized glycoprotein-specific antibodies are able to capture and isolate glycoproteins from a complex sample that is applied directly onto the microarray slides. Glycan detection then can be performed by the application of biotinylated lectins and other GBPs to the microarray slide, while binding levels can be determined using Dylight 549-Streptavidin. Through the use of an antibody panel and probing with multiple biotinylated lectins, this method allows for an effective glycosylation profile of the different proteins found in a given human or animal sample to be developed.
Introduction
Glycosylation of protein, which is the most ubiquitous post-translational modification on proteins, modifies the physical, chemical, and biological properties of a protein, and plays a fundamental role in various biological processes1-6. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases 7-12. In fact, most current cancer biomarkers, such as the L3 fraction of &alpha;-1 fetoprotein (AFP) for hepatocellular carcinoma 13-15, and CA199 for pancreatic cancer 16, 17 are all aberrant glycan moieties on glycoproteins. However, methods to study protein glycosylation have been complicated, and not suitable for routine laboratory and clinical settings. Chen et al. has recently invented a chemically blocked antibody microarray with a glycan-binding protein (GBP) detection method for high-throughput and multiplexed profile glycosylation of native glycoproteins in a complex sample 18. In this affinity based microarray method, multiple immobilized glycoprotein-specific antibodies capture and isolate glycoproteins from the complex mixture directly on the microarray slide, and the glycans on each individual captured protein are measured by GBPs. Because all normal antibodies contain N-glycans which could be recognized by most GBPs, the critical step of this method is to chemically block the glycans on the antibodies from binding to GBP. In the procedure, the cis-diol groups of the glycans on the antibodies were first oxidized to aldehyde groups by using NaIO4 in sodium acetate buffer avoiding light. The aldehyde groups were then conjugated to the hydrazide group of a cross-linker, 4-(4-N-MaleimidoPhenyl)butyric acid Hydrazide HCl (MPBH), followed by the conjugation of a dipeptide, Cys-Gly, to the maleimide group of the MPBH. Thus, the cis-diol groups on glycans of antibodies were converted into bulky none hydroxyl groups, which hindered the lectins and other GBPs bindings to the capture antibodies. This blocking procedure makes the GBPs and lectins bind only to the glycans of captured proteins. After this chemically blocking, serum samples were incubated with the antibody microarray, followed by the glycans detection by using different biotinylated lectins and GBPs, and visualized with Cy3-streptavidin. The parallel use of an antibody panel and multiple lectin probing provides discrete glycosylation profiles of multiple proteins in a given sample 18-20. This method has been used successfully in multiple different labs 1, 7, 13, 19-31. However, stability of MPBH and Cys-Gly, complicated and extended procedure in this method affect the reproducibility, effectiveness and efficiency of the method. In this new protocol, we replaced both MPBH and Cys-Gly with one much more stable reagent glutamic acid hydrazide (Glu-hydrazide), which significantly improved the reproducibility of the method, simplified and shorten the whole procedure so that the it can be completed within one working day. In this new protocol, we describe the detailed procedure of the protocol which can be readily adopted by normal labs for routine protein glycosylation study and techniques which are necessary to obtain reproducible and repeatable results.

Watch this Scientific Journal Video about Chemically-blocked Antibody Microarray for Multiplexed High-throughput Profiling of Specific Protein Glycosylation in Complex Samples at JoVE.com

Chemically-blocked Antibody Microarray for Multiplexed High-throughput Profiling of Specific Protein Glycosylation in Complex Samples

In this study, we describe an improved protocol for a multiplexed high-throughput antibody microarray with lectin detection method that can be used in glycosylation profiling of specific proteins. This protocol features new reliable reagents and significantly reduces the time, cost, and lab equipment requirements as compared to the previous procedure.

 In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that ...

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Research

JoVE Journal

Biology

Antibody Profiling by Luciferase Immunoprecipitation Systems (LIPS)

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease mechanisms and may elucidate new markers to substratify disease with different clinical features and better understand pathogenesis. We have developed a highly quantitative method called Luciferase Immunoprecipitation Systems (LIPS) for profiling patient sera antibody responses to autoantigens and pathogen antigens associated with infection. Unlike ELISAs, the highly sensitive LIPS is easily implemented to survey humoral serological response profiles to different antigens in a universal format and produces dynamic antibody titer ranges up to 6 log10 for some antigens. In these studies, quantitative profiling by LIPS of patient humoral responses against panels of antigens or even the entire proteome of some pathogens (i.e. HIV), is typically more informative than testing a single antigen by ELISA. In addition, LIPS also eliminates time and effort needed to produce highly purified antigens as well as the labor-intensive assay optimization steps needed for standard ELISAs. Here we provide a detailed protocol describing the technical aspects of performing LIPS assays for readily profiling antibody responses to single or multiple antigens.

Antibody Profiling by Luciferase Immunoprecipitation Systems LIPS

The technical aspects of performing LIPS (Luciferase Immunoprecipitation Systems) are described. The overall approach involves expressing chimeric genes encoding antigens fused to Renilla luciferase (Ruc) in mammalian cells. Crude Ruc-antigen extracts are then prepared and, without purification, employed in immunoprecipitation assays to quantify antibodies.

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease ...

High-Throughput Measurement and Classification of Organic P in Environmental Samples

Many types of organic phosphorus (P) molecules exist in environmental samples1. Traditional P measurements do not detect these organic P compounds since they do not react with colorimetric reagents2,3. Enzymatic hydrolysis (EH) is an emerging method for accurately characterizing organic P forms in environmental samples4,5. This method is only trumped in accuracy by Phosphorus-31 Nuclear Magnetic Resonance Spectroscopy (31P-NMR) -a method that is expensive and requires specialized technical training6. We have adapted an enzymatic hydrolysis method capable of measuring three classes of phosphorus (monoester P, diester P and inorganic P) to a microplate reader system7. This method provides researchers with a fast, accurate, affordable and user-friendly means to measure P species in soils, sediments, manures and, if concentrated, aquatic samples. This is the only high-throughput method for measuring the forms and enzyme-lability of organic P that can be performed in a standard laboratory. The resulting data provides insight to scientists studying system nutrient content and eutrophication potential.

This protocol describes a high-throughput method of enzymatic hydrolysis that utilizes a microplate reader to measure and classify soil phosphorus as P monoesters, P diesters and inorganic P. Up to 96 samples can be measured at one time in a standard laboratory.

Many types of organic phosphorus (P) molecules exist in environmental samples1.  Traditional P measurements do not detect these organic P compounds since ...

Identification and Characterization of Protein Glycosylation using Specific Endo- and Exoglycosidases

Glycosylation, the addition of covalently linked sugars, is a major post-translational modification of proteins that can significantly affect processes such as cell adhesion, molecular trafficking, clearance, and signal transduction1-4. In eukaryotes, the most common glycosylation modifications in the secretory pathway are additions at consensus asparagine residues (N-linked); or at serine or threonine residues (O-linked) (Figure 1). Initiation of N-glycan synthesis is highly conserved in eukaryotes, while the end products can vary greatly among different species, tissues, or proteins. Some glycans remain unmodified ("high mannose N-glycans") or are further processed in the Golgi ("complex N-glycans"). Greater diversity is found for O-glycans, which start with a common N-Acetylgalactosamine (GalNAc) residue in animal cells but differ in lower organisms1.
The detailed analysis of the glycosylation of proteins is a field unto itself and requires extensive resources and expertise to execute properly. However a variety of available enzymes that remove sugars (glycosidases) makes possible to have a general idea of the glycosylation status of a protein in a standard laboratory setting. Here we illustrate the use of glycosidases for the analysis of a model glycoprotein: recombinant human chorionic gonadotropin beta (hCG&beta;), which carries two N-glycans and four O-glycans 5. The technique requires only simple instrumentation and typical consumables, and it can be readily adapted to the analysis of multiple glycoprotein samples.
Several enzymes can be used in parallel to study a glycoprotein. PNGase F is able to remove almost all types of N-linked glycans6,7. For O-glycans, there is no available enzyme that can cleave an intact oligosaccharide from the protein backbone. Instead, O-glycans are trimmed by exoglycosidases to a short core, which is then easily removed by O-Glycosidase. The Protein Deglycosylation Mix contains PNGase F, O-Glycosidase, Neuraminidase (sialidase), &beta;1-4 Galactosidase, and &beta;-N-Acetylglucosaminidase. It is used to simultaneously remove N-glycans and some O-glycans8 . Finally, the Deglycosylation Mix was supplemented with a mixture of other exoglycosidases (&alpha;-N-Acetylgalactosaminidase, &alpha;1-2 Fucosidase, &alpha;1-3,6 Galactosidase, and &beta;1-3 Galactosidase ), which help remove otherwise resistant monosaccharides that could be present in certain O-glycans.
SDS-PAGE/Coomasie blue is used to visualize differences in protein migration before and after glycosidase treatment. In addition, a sugar-specific staining method, ProQ Emerald-300, shows diminished signal as glycans are successively removed. This protocol is designed for the analysis of small amounts of glycoprotein (0.5 to 2 &mu;g), although enzymatic deglycosylation can be scaled up to accommodate larger quantities of protein as needed.

Using specific glycosidases to remove sugars from glycoproteins followed by SDS-PAGE is a valuable method to detect glycan modifications on protein samples and is a good choice for initial glycobiology studies. Changes following deglycosylation can be detected as shifts in gel mobility or by staining with glycan sensitive reagents.

Glycosylation, the addition of covalently linked sugars, is a major post-translational modification of proteins that can significantly affect processes ...

Recovery of Adult Zebrafish Hearts for High-throughput Applications

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and purification of RNA, DNA, and proteins. All of these applications demand the rapid recovery of significant numbers of zebrafish hearts to avoid gene regulatory, metabolic, and other changes that begin after death. Adult zebrafish hearts are also required for studying heart structure for a variety of mutants and for studying heart regeneration. However, the traditional zebrafish heart dissection is slow and difficult and requires specialized tools, making large-scale dissection of adult zebrafish hearts tedious. Traditional methods also harbor the risk of damaging the heart during the dissection. Here, we describe a method for dissection of adult zebrafish hearts that is fast, reproducible, and preserves heart architecture. Furthermore, this method does not require specialized tools, is painless for the zebrafish, can be performed on fresh or fixed specimens, and can be performed on zebrafish as young as one month old. The approach described expands the use of adult zebrafish for cardiovascular research.

Use of zebrafish for cardiovascular research is expanding towards research on adult hearts. For these applications, quick and simple isolation of cardiac tissues is key to avoid post-mortem changes and to obtain an adequate number of samples. Here, we describe a fast and reproducible method for dissecting adult zebrafish hearts.

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

Quantitation of Protein Expression and Co-localization Using Multiplexed Immuno-histochemical Staining and Multispectral Imaging

Immunohistochemistry is a commonly used clinical and research lab detection technique for investigating protein expression and localization within tissues. Many semi-quantitative systems have been developed for scoring expression using immunohistochemistry, but inherent subjectivity limits reproducibility and accuracy of results. Furthermore, the investigation of spatially overlapping biomarkers such as nuclear transcription factors is difficult with current immunohistochemistry techniques. We have developed and optimized a system for simultaneous investigation of multiple proteins using high throughput methods of multiplexed immunohistochemistry and multispectral imaging. Multiplexed immunohistochemistry is performed by sequential application of primary antibodies with secondary antibodies conjugated to horseradish peroxidase or alkaline phosphatase. Different chromogens are used to detect each protein of interest. Stained slides are loaded into an automated slide scanner and a protocol is created for automated image acquisition. A spectral library is created by staining a set of slides with a single chromogen on each. A subset of representative stained images are imported into multispectral imaging software and an algorithm for distinguishing tissue type is created by defining tissue compartments on images. Subcellular compartments are segmented by using hematoxylin counterstain and adjusting the intrinsic algorithm. Thresholding is applied to determine positivity and protein co-localization. The final algorithm is then applied to the entire set of tissues. Resulting data allows the user to evaluate protein expression based on tissue type (ex. epithelia vs. stroma) and subcellular compartment (nucleus vs. cytoplasm vs. plasma membrane). Co-localization analysis allows for investigation of double-positive, double-negative, and single-positive cell types. Combining multispectral imaging with multiplexed immunohistochemistry and automated image acquisition is an objective, high-throughput method for investigation of biomarkers within tissues.

Immunohistochemistry is a powerful lab technique for evaluating protein localization and expression within tissues. Current semi-automated methods for quantitation introduce subjectivity and often create irreproducible results. Herein, we describe methods for multiplexed immunohistochemistry and objective quantitation of protein expression and co-localization using multispectral imaging.

Immunohistochemistry is a commonly used clinical and research lab detection technique for investigating protein expression and localization within ...

A Protocol for Functional Assessment of Whole-Protein Saturation Mutagenesis Libraries Utilizing High-Throughput Sequencing

Site-directed mutagenesis has long been used as a method to interrogate protein structure, function and evolution. Recent advances in massively-parallel sequencing technology have opened up the possibility of assessing the functional or fitness effects of large numbers of mutations simultaneously. Here, we present a protocol for experimentally determining the effects of all possible single amino acid mutations in a protein of interest utilizing high-throughput sequencing technology, using the 263 amino acid antibiotic resistance enzyme TEM-1 &#946;-lactamase as an example. In this approach, a whole-protein saturation mutagenesis library is constructed by site-directed mutagenic PCR, randomizing each position individually to all possible amino acids. The library is then transformed into bacteria, and selected for the ability to confer resistance to &#946;-lactam antibiotics. The fitness effect of each mutation is then determined by deep sequencing of the library before and after selection. Importantly, this protocol introduces methods which maximize sequencing read depth and permit the simultaneous selection of the entire mutation library, by mixing adjacent positions into groups of length accommodated by high-throughput sequencing read length and utilizing orthogonal primers to barcode each group. Representative results using this protocol are provided by assessing the fitness effects of all single amino acid mutations in TEM-1 at a clinically relevant dosage of ampicillin. The method should be easily extendable to other proteins for which a high-throughput selection assay is in place.

We present a protocol for the functional assessment of comprehensive single-site saturation mutagenesis libraries of proteins utilizing high-throughput sequencing. Importantly, this approach uses orthogonal primer pairs to multiplex library construction and sequencing. Representative results using TEM-1 &#946;-lactamase selected at a clinically relevant dosage of ampicillin are provided.

Site-directed mutagenesis has long been used as a method to interrogate protein structure, function and evolution. Recent advances in massively-parallel ...

High-throughput Screening for Protein-based Inheritance in S. cerevisiae

The encoding of biological information that is accessible to future generations is generally achieved via changes to the DNA sequence. Long-lived inheritance encoded in protein conformation (rather than sequence) has long been viewed as paradigm-shifting but rare. The best characterized examples of such epigenetic elements are prions, which possess a self-assembling behavior that can drive the heritable manifestation of new phenotypes. Many archetypal prions display a striking N/Q-rich sequence bias and assemble into an amyloid fold. These unusual features have informed most screening efforts to identify new prion proteins. However, at least three known prions (including the founding prion, PrPSc) do not harbor these biochemical characteristics. We therefore developed an alternative method to probe the scope of protein-based inheritance based on a property of mass action: the transient overexpression of prion proteins increases the frequency at which they acquire a self-templating conformation. This paper describes a method for analyzing the capacity of the yeast ORFeome to elicit protein-based inheritance. Using this strategy, we previously found that &gt;1% of yeast proteins could fuel the emergence of biological traits that were long-lived, stable, and arose more frequently than genetic mutation. This approach can be employed in high throughput across entire ORFeomes or as a targeted screening paradigm for specific genetic networks or environmental stimuli. Just as forward genetic screens define numerous developmental and signaling pathways, these techniques provide a methodology to investigate the influence of protein-based inheritance in biological processes.

High-throughput Screening for Protein-based Inheritance in S. cerevisiae

This protocol describes a high-throughput methodology to functionally screen for protein-based inheritance in S. cerevisiae.

The encoding of biological information that is accessible to future generations is generally achieved via changes to the DNA sequence. Long-lived ...

TChIP-Seq: Cell-Type-Specific Epigenome Profiling

Epigenetic regulation plays central roles in gene expression. Since histone modification was discovered in the 1960s, its physiological and pathological functions have been extensively studied. Indeed, the advent of next-generation deep sequencing and chromatin immunoprecipitation (ChIP) via specific histone modification antibodies has revolutionized our view of epigenetic regulation across the genome. Conversely, tissues typically consist of diverse cell types, and their complex mixture poses analytic challenges to investigating the epigenome in a particular cell type. To address the cell type-specific chromatin state in a genome-wide manner, we recently developed tandem chromatin immunoprecipitation sequencing (tChIP-Seq), which is based on the selective purification of chromatin by tagged core histone proteins from cell types of interest, followed by ChIP-Seq. The goal of this protocol is the introduction of best practices of tChIP-Seq. This technique provides a versatile tool for tissue-specific epigenome investigation in diverse histone modifications and model organisms.

We describe a step-by-step protocol for tandem chromatin immunoprecipitation sequencing (tChIP-Seq) that enables the analysis of cell-type-specific genome-wide histone modification.

Epigenetic regulation plays central roles in gene expression. Since histone modification was discovered in the 1960s, its physiological and pathological ...