Este trabalho foi financiado pelo Instituto de Pesquisa de Vírus Hepatite e.

<ol>
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1. Proteína alvo e seleção anticorpo de captura Antes do ensaio microarray anticorpo, alguns reagentes e materiais são necessários para ser considerado e preparado. Para conceber um microarray anticorpo para glicano perfil ou rastreio de glicano biomarcador, um painel de anticorpos específicos para os candidatos de glicoproteína deve ser determinada de acordo com a literatura ou a partir dos resultados anteriores. Esses anticorpos foram normalmente compradas de fornecedores diferentes, t...

<table border="1"><tbody><tr><td> ID </td><td> Nome do reagente </td><td> Abreviatura </td><td> Companhia </td><td> Catalogo </td></tr><tr><td> L1 </td><td> Concanavalina A biotinilado </td><td> ConA </td><td> Vector Laboratories </td><td> BK-1000 </td></tr><tr><td> L2 </td><td> Biotinilado Sambucus Nigra Lectina </td><td> SNA </td><td> Vector Laboratories </td><td> B-1305 </td></tr><tr><td> L3 </td><td> Biotinilado Lens culinaris aglutinina </td><td> LCA </td><td> Vector Laboratories </td><td> BK-2000 </td></tr><tr><td> L4 </td><td> Biotinilado Ricinus communis aglutinina eu </td><td> RCA </td><td> Vector Laboratories </td><td> BK-1000 </td></tr><tr><td> L5 </td><td> Biotinilado Aleuria Lectina Aurantia </td><td> AAL </td><td> Vector Laboratories </td><td> B-1395 </td></tr><tr><td> L6 </td><td> Biotinilado Erythrina Lectina Cristagalli </td><td> ECL </td><td> Vector Laboratories </td><td> BK-3000 </td></tr><tr><td> L7 </td><td> Biotinilado Griffonia (Bandeiraea) Lectina Simplicifolia II </td><td> GSL II </td><td> Vector Laboratories </td><td> BK-3000 </td></tr><tr><td> L8 </td><td> Biotinilado Gérmen de Trigo aglutinina </td><td> WGA </td><td> Vector Laboratories </td><td> BK-1000 </td></tr><tr><td> L9 </td><td> Erythroagglutinin vulgaris biotinilado Phaseolus </td><td> PHA-E </td><td> Vector Laboratories </td><td> BK-2000 </td></tr><tr><td> L10 </td><td> Leucoagglutinin vulgaris biotinilado Phaseolus </td><td> PHA-L </td><td> Vector Laboratories </td><td> BK-2000 </td></tr><tr><td> L11 </td><td> Aglutinina de amendoim biotinilado </td><td> PNA </td><td> Vector Laboratories </td><td> BK-1000 </td></tr><tr><td> L12 </td><td> Biotinilado Pisum sativum aglutinina </td><td> PSA </td><td> Vector Laboratories </td><td> BK-2000 </td></tr><tr><td> L13 </td><td> Biotinilado Dolichos biflorus aglutinina </td><td> DBA </td><td> Vector Laboratories </td><td> BK-1000 </td></tr><tr><td> L14 </td><td> Lectina Stramonium biotinilado Datura </td><td> DSL </td><td> Vector Laboratories </td><td> BK-3000 </td></tr><tr><td> L15 </td><td> Aglutinina Sophora Japonica biotinilado </td><td> SJA </td><td> Vector Laboratories </td><td> BK-2000 </td></tr><tr><td> L16 </td><td> Aglutinina de soja biotinilado </td><td> SBA </td><td> Vector Laboratories </td><td> BK-1000 </td></tr><tr><td> L17 </td><td> Biotinilado Solanum tuberosum (Batata) Lectina </td><td> STL </td><td> Vector Laboratories </td><td> BK-3000 </td></tr><tr><td> L18 </td><td> Biotinilado Griffonia (Bandeiraea) Simplicifolia Lectina I </td><td> Eu GSL </td><td> Vector Laboratories </td><td> BK-2000 </td></tr><tr><td> L19 </td><td> Biotinilado Vicia Lectina Villosa </td><td> VVL </td><td> Vector Laboratories </td><td> BK-2000 </td></tr><tr><td> L20 </td><td> Biotinilado Lycopersicon esculentum (tomate) Lectina </td><td> LEL </td><td> Vector Laboratories </td><td> BK-3000 </td></tr><tr><td> L21 </td><td> Ulex biotinilado Europaeus aglutinina eu </td><td> UEA I </td><td> Vector Laboratories </td><td> BK-1000 </td></tr><tr><td> L22 </td><td> Jacalina biotinilado </td><td> Jacalina </td><td> Vector Laboratories </td><td> BK-3000 </td></tr><tr><td> A1 </td><td> F cabra (ab &#39;) 2 Fragmento IgM anti-humana, o anticorpo Fc5μ </td><td> IgM </td><td> Jackson Immuno Research </td><td> 109-006-129 </td></tr><tr><td> A2 </td><td> Burro F (ab &#39;) 2 Frag anti-IgG humana (H + L) umtibody </td><td> AB1 </td><td> Jackson Immuno Research </td><td> 709-006-149 </td></tr><tr><td> A3 </td><td> De ratinho anti-IgG humana F (ab &#39;) 2 de anticorpo monoclonal </td><td> AB3 </td><td> Jackson Immuno Research </td><td> 209-005-097 </td></tr><tr><td> A4 </td><td> Cabra anti-humano alfa 2 macroglobulina anticorpo policlonal </td><td> A2M </td><td> GeneTex </td><td> GTX62924 </td></tr><tr><td> A5 </td><td> De coelho anti-anticorpo humano alfa-1-antitripsina policlonal </td><td> A1AT </td><td> Lee Biosiences </td><td> CA1T-80A </td></tr><tr><td> A6 </td><td> De ratinho anti-anticorpo humano alfa-1-antitripsina monoclonal </td><td> A1AT </td><td> Sigma Aldrich </td><td> SAB4200198 </td></tr><tr><td> A7 </td><td> De coelho anti-anticorpo humano alfa-1-antitripsina policlonal </td><td> ACT </td><td> Neomarkers </td><td> RB-367-A1 </td></tr><tr><td> A8 </td><td> De coelho anti-humanoalfa-1-antiquimotripsina anticorpo policlonal </td><td> ACT </td><td> Fisher Scientific </td><td> RB9213R7 </td></tr><tr><td> A9 </td><td> Rato anti-humana de anticorpo monoclonal de transferrina </td><td> Transferrina </td><td> GeneTex </td><td> GTX101035 </td></tr><tr><td> A10 </td><td> De coelho anti-anticorpo humano policlonal de transferrina </td><td> Transferrina </td><td> GeneTex </td><td> GTX77130 </td></tr><tr><td> A11 </td><td> Cabra anti-humano apolipoproteína anticorpo policlonal J </td><td> ApoJ </td><td> Abcam </td><td> ab7610 </td></tr><tr><td> A12 </td><td> Rato anti-humana de GP73 anticorpo monoclonal </td><td> GP73 </td><td> Abbott </td><td> 14H4-23 </td></tr><tr><td> A13 </td><td> Rato anti-humana de GP73 anticorpo monoclonal </td><td> GP73 </td><td> Santa Cruz Biotechnology, Inc. </td><td> sc-101.275 </td></tr><tr><td> A14 </td><td> De coelho anti-humano de alfa-1 fetoprotein anticorpo policlonal </td><td> AFP </td><td> Genway </td><td> GWB-41C966 </td></tr><tr><td> A15 </td><td> De ratinho anti-anticorpo humano de alfa-1 fetoproteína monoclonal </td><td> AFP </td><td> Fitzgerald </td><td> 10-A05A </td></tr><tr><td> A16 </td><td> Rato anti-humana de anticorpo monoclonal hemopexina </td><td> Hemopexina </td><td> Assaypro </td><td> 60190-05011 </td></tr><tr><td> A17 </td><td> Rato anti-humano glypican-3 (1G12) anticorpo monoclonal </td><td> GPL3 </td><td> Santa Cruz Bio </td><td> sc-65.443 </td></tr><tr><td> A18 </td><td> Rato anti-humano Cininogênio anticorpo (LMW) monoclonal </td><td> Cininogênio </td><td> Assaypro </td><td> 20333-05011 </td></tr><tr><td> A19 </td><td> De coelho anti-anticorpo humano MMP-21 monoclonal </td><td> MMP21 </td><td> Epitomic </td><td> 1955-1 </td></tr><tr><td> A20 </td><td> Rato anti-humano CEACAM-1 anticorpos monoclonaisy </td><td> CEACAM </td><td> R &amp; D Systems </td><td> MAB1180 </td></tr><tr><td> A21 </td><td> Rat anti-humano DPPIV/CD26 anticorpo monoclonal </td><td> DPPIV </td><td> R &amp; D Systems </td><td> MAB22441 </td></tr><tr><td> A22 </td><td> Rato anti-humano PIVKA anticorpo monoclonal II </td><td> PIVICA </td><td> Cristal chem </td><td> 8040 </td></tr><tr><td> A23 </td><td> Antigénio de ratinho anti-carcinoembriónico </td><td> CEA </td><td> EUA biológica </td><td> C1300 </td></tr><tr><td> A24 </td><td> Antigen de ratinho anti-CA125 Câncer </td><td> CA125 </td><td> EUA biológica </td><td> C0050-01D </td></tr><tr><td> A25 </td><td> Antigénio de ratinho anti-CA19-9 Câncer </td><td> CA19-9 </td><td> EUA biológica </td><td> C0075-18 </td></tr><tr><td> A26 </td><td> De ratinho anti-Lewis anticorpo monoclonal x </td><td> Lewis X </td><td> Calbiochem </td><td> 434631 </td></tr><tr><td> bio </td><td> Biotinilado BSA (controle positivo) </td><td> Bio </td><td> Home-made </td><td> N / A </td></tr></tbody></table> Lista Tabela 1. De lectinas e anticorpos utilizados neste protocolo. <table border="1"><tbody><tr><td> Nome do reagente s / equipamentos </td><td> Companhia </td><td> Número de catálogo </td></tr><tr><td> Microarrayer sem contato </td><td> BioDot Inc </td><td> sciFLEXARRAYER </td></tr><tr><td> 384 microplacas </td><td> Pescador </td><td> 14-230-243 </td></tr><tr><td> FoodSaver </td><td> FoodSaver </td><td> V3835 </td></tr><tr><td> Nitrocelulose Ultrathin Coate microarray desliza </td><td> Gentel </td><td> PATH </td></tr><tr><td> Deslize Imprinter (opcional) </td><td> A Companhia Gel </td><td> WSP60-1 </td></tr><tr><td> Sacudidor </td><td> Pescador </td><td> 15-453-211 </td></tr><tr><td> Centrifugar </td><td> Eppendorf </td><td> 5804 000.013 </td></tr><tr><td> Deslize prato de coloração de lavagem bacia / Slide com rack removível </td><td> Pescador </td><td> 08-812 </td></tr><tr><td> Deslize incubação caixa de lâminas câmara / microscópio </td><td> Pescador </td><td> 03-448-5 </td></tr><tr><td> Brij 35, 30% de solução w / v em água </td><td> Acros Organics </td><td> AC32958-0025 </td></tr><tr><td> Tween-20 </td><td> Pescador </td><td> P337-100 </td></tr><tr><td> Periodato de sódio (NaIO4) </td><td> Sigma </td><td> 311448 </td></tr><tr><td> Ácido L-glutâmico γ-hidrazida </td><td> Sigma </td><td> G-7257 </td></tr><tr><td> De acetato de sódio anidro (CH 3 COONa) </td><td> Sigma </td><td> S2889 </td></tr><tr><td> Albumina de soro bovino (BSA) </td><td> Lampire Biológica Labs </td> &lt;td&gt; 7500804 </td></tr><tr><td> Tampão fosfato salino (PBS) (10X) </td><td> Denville Científico </td><td> CP4390-48 </td></tr><tr><td> Dylight NeutrAvidin 549 conjugado </td><td> Thermo </td><td> 22837 </td></tr><tr><td> Protease Comprimidos Coquetel Inibidor </td><td> Roche </td><td> 4693159001 </td></tr><tr><td> ChromPure IgG humana, fragmento Fc </td><td> Jackson Immunoresearch </td><td> 009-000-008 </td></tr><tr><td> ChromPure IgG humana, molécula inteira </td><td> Jackson Immunoresearch </td><td> 009-000-003 </td></tr><tr><td> ChromPure IgG de rato, molécula inteira </td><td> Jackson Immunoresearch </td><td> 015-000-003 </td></tr><tr><td> ChromPure IgG de rato, fragmento Fe </td><td> Jackson Immunoresearch </td><td> 015-000-008 </td></tr><tr><td> ChromPure IgG de coelho, molécula inteira </td><td> Jackson Immunoresearch </td><td> 011-000-003 </td></tr><tr><td> ChromPure Donkey IgG, molécula inteira </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> Lista Tabela 2. De equipamentos e reagentes utilizados neste protocolo.

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

Neste estudo, nós descrevemos um protocolo melhorado para um microarray anticorpo multiplexado de alto rendimento com o método de detecção de lectina que pode ser usado em perfil de glicosilação de proteínas específicas. Este protocolo apresenta novos reagentes fiáveis ​​e reduz significativamente o tempo, custo e requisitos de equipamento de laboratório, em comparação com o procedimento anterior.

Microarray de anticorpos quimicamente bloqueada para Multiplexed perfil de alta capacidade de glicosilação proteína específica em amostras complexas

 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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JoVE Journal

Biology

15.3K visualizações.  Institute for Hepatitis and Virus Research. Neste estudo, nós descrevemos um protocolo melhorado para um microarray anticorpo multiplexado de alto rendimento com o método de detecção de lectina que pode ser usado em perfil de glicosilação de proteínas específicas. Este protocolo apresenta novos reagentes fiáveis ​​e reduz significativamente o tempo, custo e requisitos de equipamento de laboratório, em comparação com o procedimento anterior.

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

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.

Perfil de anticorpos por sistemas de imunoprecipitação luciferase (LIPS)

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

Biologia

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.

High-Throughput medição e classificação de P orgânico em amostras ambientais

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.

Identificação e caracterização de glicosilação de proteínas específicas usando endo-e exoglycosidases

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

Flow Cytometric Analysis of Bimolecular Fluorescence Complementation: A High Throughput Quantitative Method to Study Protein-protein Interaction

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC is based on the production of fluorescence using two non-fluorescent fragments of a fluorescent protein (Venus, a Yellow Fluorescent Protein variant, is used here). Non-fluorescent Venus fragments (VN and VC) are fused to two interacting proteins (in this case, AKAP-Lbc and PDE4D3), yielding fluorescence due to VN-AKAP-Lbc-VC-PDE4D3 interaction and the formation of a functional fluorescent protein inside cells.
BiFC provides information on the subcellular localization of protein complexes and the strength of protein interactions based on fluorescence intensity. However, BiFC analysis using microscopy to quantify the strength of protein-protein interaction is time-consuming and somewhat subjective due to heterogeneity in protein expression and interaction. By coupling flow cytometric analysis with BiFC methodology, the fluorescent BiFC protein-protein interaction signal can be accurately measured for a large quantity of cells in a short time. Here, we demonstrate an application of this methodology to map regions in PDE4D3 that are required for the interaction with AKAP-Lbc. This high throughput methodology can be applied to screening factors that regulate protein-protein interaction.

Flow cytometric analysis of Bimolecular Fluorescence Complementation provides a high throughput quantitative method to study protein-protein interaction. This methodology can be applied to mapping protein binding sites and for screening factors that regulate protein-protein interaction.

Citometria de fluxo e de Complementação de fluorescência Bimolecular: A alta taxa de transferência método quantitativo para estudo de interação proteína-proteína

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC ...

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.

Recuperação de Zebrafish Adulto Corações para aplicações de alto rendimento

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.

Verde Fluorescente à base de proteínas Expressão Rastreio de Proteínas de Membrana em<em&gt; Escherichia coli</em

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.

Rastreio de alto rendimento para moduladores químicos de Genes Post-transcriptionally Regulamentados

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

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.

Um Protocolo de Avaliação Funcional da Whole-Protein saturação mutagênese Bibliotecas Utilizando-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 ...

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.

Elevado-throughput Screening para herança à base de proteínas no 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.

TChIP-Seq: Epigenoma célula tipo-específicas de criação de perfil

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