Ce travail a été soutenu par le conseil de Singapour académique de recherche (ARC) [numéro de la subvention 90/07]. Nous remercions pour le soutien radiodurans.

Le protocole de texte complet et détaillé de cette procédure expérimentale est disponible dans Current Protocols in Molecular Biology. Détaillées étape par étape les instructions pour l&#39;assemblage du sandwich de gel et pour les appareils de gel peut être trouvé sur le site BioRad 3. Équipement requis: Les plaques de verre (intérieur et extérieur) 10 cm de cellules: 10,1 x 7,3 cm (plaque interne), 10,1 x 8,2 cm (plaque extérieure), BioRad 20 cellules cm: 20 x 20 cm (plaque interne), 20 x 22,3 cm (plaque extérieure), BioRad De 0,5 à 1,5 mm de gel de peigne et entretoises Se jetant Gel Gel appareil avec le couvercle et les câbles Alimentation haute tension Bloc de chauffage ou l&#39;eau du bain Pipettes sérologiques et l&#39;aide de pipette Pipette et pointes de pipette Sèche-Gel ou un scanner Réactifs et solutions: Urée (ultra-pure) Solution de polyacrylamide 40% (29:1) 10 x TBE solution (Tris-Borate, EDTA) Déminéralisée, eau distillée TEMED 30% (p / v) solution de persulfate d&#39;ammonium 0,5 x TBE solution Formamide EDTA Cyanol Xylène Bleu de bromophénol Méthanol Éthanol <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> Volume </td><td colspan="3" style="text-align:center;"> 50 ml </td><td colspan="3" style="text-align:center;"> 60 ml </td><td colspan="3" style="text-align:center;"> 10 ml </td></tr><tr><td> Concentration de l&#39;acrylamide </td><td> 10% </td><td> 12,5% </td><td> 15% </td><td> 10% </td><td> 12,5% </td><td> 15% </td><td> 10% </td><td> 12,5% </td><td> 15% </td></tr><tr><td> g d&#39;urée </td><td> 24 </td><td> 24 </td><td> 24 </td><td> 28,8 </td><td> 28,8 </td><td> 28,8 </td><td> 4.8 </td><td> 4.8 </td><td> 4.8 </td></tr><tr><td> ml à 40% Acryl (29:1) </td><td> 12,5 </td><td> 15.625 </td><td> 18,75 </td><td> 15 </td><td> 18,75 </td><td> 22,5 </td><td> 2.5 </td><td> 3,125 </td><td> 3,75 </td></tr><tr><td> APS ul 30% </td><td> 166 </td><td> 166 </td><td> 166 </td><td> 199,2 </td><td> 199,2 </td><td> 199,2 </td><td> 33 </td><td> 33 </td><td> 33 </td></tr><tr><td> ul TEMED </td><td> 20 </td><td> 20 </td><td> 20 </td><td> 24 </td><td> 24 </td><td> 24 </td><td> 4 </td><td> 4 </td><td> 4 </td></tr><tr><td> 10 x TBE </td><td> 5 </td><td> 5 </td><td> 5 </td><td> 6 </td><td> 6 </td><td> 6 </td><td> 1 </td><td> 1 </td><td> 1 </td></tr><tr><td colspan="10"> Remplissez le volume avec déminéralisée, eau distillée </td></tr></tbody></table> Tableau 1: Réactifs et solutions nécessaires pour les diverses concentrations d&#39;acrylamide et les volumes pour résoudre seule oligonucléotides bloqués Assemblage en sandwich de gel et gel préparation <ol><li> Assemblez le gel selon la description des fabricants et de fixer le gel dans la chambre de coulée de gel 3. Utiliser 0,5-1,5 mm d&#39;épaisseur des entretoises. </li><li> Préparer la solution de polyacrylamide appropriée, conformément aux protocoles actuels de la biologie moléculaire ou en tant que figurant dans le tableau 1. Pour un gel d&#39;acrylamide dénaturant de 20 cm x 22 cm x 1,5 mm, 60 ml de solution de gel et d&#39;une 10,1 x 8,2 cm x 1 mm de gel de 5 ml solution de gel est suffisante. Agrandir les gels sont utilisés lorsque les produits attendus / bandes sont dans la fourchette de quelques bases, puis un gel de plus permettra de résoudre les bandes avec une différence d&#39;un seul nucléotide. Si les bandes attendus diffèrent dans plus de 10 nucléotides, les petits gels seront adaptés pour résoudre les fragments. Choisissez le pourcentage de polyacrylamide qui convient à vos besoins de séparation, une concentration plus élevée de polyacrylamide résoudra bas poids moléculaire des fragments. Utilisez l&#39;urée ultrapure et mélanger avec la quantité désirée de l&#39;acrylamide. Ajouter tampon TBE au mélange de gel pour obtenir une concentration finale de 0,5-1 x TBE et de remplir le volume avec déminéralisée, eau distillée. Chauffer la solution pendant 20 secondes au micro-ondes et mélangez délicatement. Pour les volumes de gel plus répéter cette étape jusqu&#39;à ce que la solution est la main chaude. </li><li> Verser le gel immédiatement à l&#39;aide d&#39;une pipette sérologique et une aide pipette automatique entre les deux plaques de verre. Évitez d&#39;introduire des bulles d&#39;air. Insérer le peigne et laisser le gel polymériser pendant 30-60 minutes. </li></ol> Mettre en place l&#39;appareil d&#39;électrophorèse et le gel prerun <ol><li> Démonter le gel de la chambre de coulée et l&#39;assemblage de l&#39;appareil gel selon les instructions du fabricant 3. </li><li> Remplir le tampon inférieure chambre tampon d&#39;esprit de fonctionnement (0,5-1 x TBE) que les plaques de verre seront sousfusionnées 2-3 cm avec le tampon. Remplir la chambre tampon supérieure au sommet du gel avec courir tampon. </li><li> Retirer délicatement le peigne et rincer les puits avec du tampon fonctionne, en utilisant une pipette et un gel des conseils de chargement. </li><li> Fixez le couvercle du système de gel et de brancher les câbles à une alimentation haute tension. Avant que vous pouvez charger vos échantillons, vous devez prerun le gel pendant au moins 30 minutes pour chauffer le gel et pour éliminer l&#39;urée reste du gel. La température optimale doit être comprise entre 45-55 ° C. Évitez les températures supérieures à 60 ° C comme les bandes pouvaient frottis ou les plaques de verre pourrait se fissurer. Choisissez watts constante pour la prerun (15-25 W par gel). </li></ol> La préparation des échantillons <ol><li> En attendant de préparer vos échantillons. Par conséquent, ajouter la quantité appropriée de 2 mix x chargement de gel à votre échantillon. Le mélange de chargement contient généralement formamide 90%, 0,5% d&#39;EDTA, 0,1% cyanol xylène et 0,1% bleu de bromophénol. </li><li> Avant les échantillons peuvent être chargés sur le gel, les échantillons doivent être dénaturée par la chaleur en chauffant les échantillons entre 70-90 ° C pendant quelques minutes. </li></ol> Charger et exécuter le gel <ol><li> Lorsque le prerun est terminée, retirez le couvercle et rincez soigneusement les poches, comme décrit précédemment que l&#39;urée est lessivé dans les puits. </li><li> Appliquer les échantillons soigneusement au fond de la poche. Évitez d&#39;introduire des bulles d&#39;air. Chargement du tampon doit être appliquée aux poches vides pour maintenir des conditions d&#39;égalité pendant la course. </li><li> Assemblez le couvercle et lancez le gel à watts constants pour maintenir une température de gel de 55 ° C similaire à la prerun. Observez la migration des colorants marqueur jusqu&#39;à ce que le front a atteint l&#39;extrémité inférieure du gel. La durée d&#39;exécution dépend du pourcentage d&#39;acrylamide utilisé, la force ionique du tampon et de l&#39;épaisseur du gel. Une course peut durer entre 2-4 heures. Vérifiez la température du gel. Un thermomètre de gel peut aider à surveiller la bonne température pendant la course. </li></ol> Traiter le gel de <ol><li> Lorsque le front de teinture a atteint la fin du gel placer le gel de la chambre inférieure du tampon. Retirer le gel de la chambre et desserrer les pinces. Retirez les entretoises et soigneusement dissimuler les plaques de verre. Si nécessaire couper les puits supérieur contenant une partie du gel. </li><li> Soigneusement transférer le gel dans un plat avec une solution de fixation du gel (même concentration de méthanol TBE, plus de 5-10% et éthanol). Laisser le gel dans la solution pendant 5-10 minutes et changer le tampon à deux reprises. </li><li> Le gel est prêt pour un traitement ultérieur en utilisant un séchoir sous vide ou de gel de numérisation directe du gel. </li></ol>

<ol>
	<li>Maniatis, T., Jeffrey, A., van deSande, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chain+length+determination+of+small+double-+and+single-stranded+DNA+molecules+by+polyacrylamide+gel+electrophoresis.">Chain length determination of small double- and single-stranded DNA molecules by polyacrylamide gel electrophoresis.</a> Biochemistry. 14, 3787-3794 (1975).</li><li>Albright, L. M., Slatko, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Denaturing+polyacrylamide+gel+electrophoresis.">Denaturing polyacrylamide gel electrophoresis.</a> Curr Protoc Nucleic Acid Chem. , (2001).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> PROTEAN II xi Cell and PROTEAN II xi 2-D Cell Instruction Manual. , (2001).</li></ol>

The authors have nothing to disclose.

<ol><li> La netteté bande dépend de plusieurs facteurs. Le volume d&#39;échantillon chargé pour les bandes pointus devrait être aussi petit que possible, c&#39;est à dire idéalement entre 1-5 pl. Toutefois, jusqu&#39;à 15 ul ne peut être chargé qui assure toujours une résolution acceptable. Moins de résultats volume d&#39;échantillon plus nettes bandes. </li><li> La qualité bande est également dépendant de l&#39;épaisseur du gel. Diluant gels, tels que 0,75 mm montrent de meilleurs résultats que 1,5 mm d&#39;épaisseur des gel...

denaturing urea polyacrylamide gel electrophoresis (urea page)

PAGE urée ou électrophorèse sur gel dénaturant de polyacrylamide urée emploie 6-8 M d&#39;urée, qui dénature l&#39;ADN secondaire ou structures d&#39;ARN et est utilisé pour leur séparation dans une matrice de gel de polyacrylamide en fonction du poids moléculaire. Fragments de 2 à 500 bases, avec des différences de longueur aussi petite que d&#39;un seul nucléotide, peuvent être séparés en utilisant cette méthode 1. La migration de l&#39;échantillon est dépendante de la concentration d&#39;acrylamide choisi. Un pourcentage plus élevé de polyacrylamide résout fragments de poids moléculaire plus faible. La combinaison de l&#39;urée et des températures de 45-55 ° C pendant la course de gel permet la séparation de l&#39;ADN non structurées ou des molécules d&#39;ARN. En général, cette méthode est nécessaire d&#39;analyser ou de purifier l&#39;ADN simple brin ou de fragments d&#39;ARN, comme les oligonucléotides synthétisés ou étiquetés ou produits à partir des réactions de clivage enzymatique. Dans cet article, vidéo, nous montre comment préparer et exécuter les gels de polyacrylamide dénaturant l&#39;urée. Conseils techniques sont inclus, en plus des 1,2 protocole original.

Watch this Scientific Journal Video about Denaturing Urea Polyacrylamide Gel Electrophoresis Urea PAGE at JoVE.com

Denaturing Urea Polyacrylamide Gel Electrophoresis Urea PAGE

Électrophorèse sur gel dénaturant de polyacrylamide urée est utilisée pour séparer l&#39;ADN simple brin ou l&#39;ARN jusqu&#39;à une limite de 500 nucléotides. L&#39;urée en combinaison avec des échantillons dénature la chaleur et non structurées brins simples migrer dans la matrice de gel en fonction de leur poids moléculaire.

Électrophorèse sur gel dénaturant Urée polyacrylamide (PAGE urée)

Urea PAGE or denaturing urea polyacrylamide gel electrophoresis employs 6-8 M urea, which denatures secondary DNA or RNA structures and is used for their ...

denaturing-urea-polyacrylamide-gel-electrophoresis-urea-page

Research

JoVE Journal

Biology

Denaturing Urea Polyacrylamide Gel Electrophoresis (Urea PAGE)

78.3K vues.  Nanyang Technological University, Singapore - NTU. Électrophorèse sur gel dénaturant de polyacrylamide urée est utilisée pour séparer l'ADN simple brin ou l'ARN jusqu'à une limite de 500 nucléotides. L'urée en combinaison avec des échantillons dénature la chaleur et non structurées brins simples migrer dans la matrice de gel en fonction de leur poids moléculaire.

Vidéo: Denaturing Urea Polyacrylamide Gel Electrophoresis Urea PAGE

Denaturing Gradient Gel Electrophoresis (DGGE)

Denaturing Gradient Gel Electrophoresis DGGE

Électrophorèse sur gel dénaturant Dégradé (DGGE)

Recherche

Biologie

Screening for Amyloid Aggregation by Semi-Denaturing Detergent-Agarose Gel Electrophoresis

Amyloid aggregation is associated with numerous protein misfolding pathologies and underlies the infectious properties of prions, which are conformationally self-templating proteins that are thought to have beneficial roles in lower organisms.  Amyloids have been notoriously difficult to study due to their insolubility and structural heterogeneity.  However, resolution of amyloid polymers based on size and detergent insolubility has been made possible by Semi-Denaturing Detergent-Agarose Gel Electrophoresis (SDD-AGE).  This technique is finding widespread use for the detection and characterization of amyloid conformational variants.  Here, we demonstrate an adaptation of this technique that facilitates its use in large-scale applications, such as screens for novel prions and other amyloidogenic proteins.  The new SDD-AGE method uses capillary transfer for greater reliability and ease of use, and allows any sized gel to be accomodated.  Thus, a large number of samples, prepared from cells or purified proteins, can be processed simultaneously for the presence of SDS-insoluble conformers of tagged proteins.

SDD-AGE is a useful technique for the detection and characterization of amyloid-like polymers in cells. Here we demonstrate an adaptation that makes this technique amenable to large-scale applications.

Le dépistage de l&#39;agrégation amyloïde par les semi-électrophorèse sur gel dénaturant Détergent-Agarose

Amyloid aggregation is associated with numerous protein misfolding pathologies and underlies the infectious properties of prions, which are ...

Fast and Sensitive Colloidal Coomassie G-250 Staining for Proteins in Polyacrylamide Gels

Coomassie Brilliant Blue (CBB) is a dye commonly used for the visualization of proteins separated by SDS-PAGE, offering a simple staining procedure and high quantitation. Furthermore, it is completely compatible with mass spectrometric protein identification. But despite these advantages, CBB is regarded to be less sensitive than silver or fluorescence stainings and therefore rarely used for the detection of proteins in analytical gel-based proteomic approaches.
Several improvements of the original Coomassie protocol1 have been made to increase the sensitivity of CBB. Two major modifications were introduced to enhance the detection of low-abundant proteins by converting the dye molecules into colloidal particles: In 1988, Neuhoff and colleagues applied 20% methanol and higher concentrations of ammonium sulfate into the CBB G-250 based staining solution2, and in 2004 Candiano et al. established Blue Silver using CBB G-250 with phosphoric acid in the presence of ammonium sulfate and methanol3. Nevertheless, all these modifications just allow a detection of approximately 10 ng protein. A widely fameless protocol for colloidal Coomassie staining was published by Kang et al. in 2002 where they modified Neuhoff's colloidal CBB staining protocol regarding the complexing substances. Instead of ammonium sulfate they used aluminum sulfate and methanol was replaced by the less toxic ethanol4. The novel aluminum-based staining in Kang's study showed superior sensitivity that detects as low as 1 ng/band (phosphorylase b) with little sensitivity variation depending on proteins.
Here, we demonstrate application of Kang's protocol for fast and sensitive colloidal Coomassie staining of proteins in analytical purposes. We will illustrate the quick and easy protocol using two-dimensional gels routinely performed in our working group.

This video shall popularize a colloidal Coomassie G-250 staining protocol according to Kang et al. for the detection of average 4 ng protein in gels. The staining is completed within 2 hours and without any effort. We routinely use Kang's protocol for analytical purposes in gel-based proteomics.

Rapide et sensible colloïdal Coomassie G-250 de coloration des protéines dans des gels de polyacrylamide

Coomassie Brilliant Blue (CBB) is a dye commonly used for the visualization of proteins separated by SDS-PAGE, offering a simple staining procedure and ...

Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) for Analysis of Multiprotein Complexes from Cellular Lysates

Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other proteins (Sali, Glaeser et al. 2003). Thus, the study of protein-protein interaction networks requires the detailed characterization of MPCs to gain an integrative understanding of protein function and regulation. For identification and analysis, MPCs must be separated under native conditions. In this video, we describe the analysis of MPCs by blue native polyacrylamide gel electrophoresis (BN-PAGE). BN-PAGE is a technique that allows separation of MPCs in a native conformation with a higher resolution than offered by gel filtration or sucrose density ultracentrifugation, and is therefore useful to determine MPC size, composition, and relative abundance (Sch&auml;gger and von Jagow 1991); (Sch&auml;gger, Cramer et al. 1994). By this method, proteins are separated according to their hydrodynamic size and shape in a polyacrylamide matrix. Here, we demonstrate the analysis of MPCs of total cellular lysates, pointing out that lysate dialysis is the crucial step to make BN-PAGE applicable to these biological samples. Using a combination of first dimension BN- and second dimension SDS-PAGE, we show that MPCs separated by BN-PAGE can be further subdivided into their individual constituents by SDS-PAGE. Visualization of the MPC components upon gel separation is performed by standard immunoblotting. As an example for MPC analysis by BN-PAGE, we chose the well-characterized eukaryotic 19S, 20S, and 26S proteasomes.

Blue Native Polyacrylamide Gel Electrophoresis BN-PAGE for Analysis of Multiprotein Complexes from Cellular Lysates

In this video, we describe the characterization of multiprotein complexes (MPCs) by blue native polyacrylamide gel electrophoresis (BN-PAGE). In a first dimension, dialyzed cellular lysates are separated by BN-PAGE to identify individual MPCs. In a second dimension SDS-PAGE, MPCs of interest are further subdivided to analyze their constituents by immunoblotting.

Bleu d&#39;électrophorèse sur gel de polyacrylamide natif (BN-PAGE) pour l&#39;analyse des complexes multiprotéiques de lysats cellulaires

Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other ...

Agarose Gel Electrophoresis for the Separation of DNA Fragments

Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from 100 bp to 25 kb1. Agarose is isolated from the seaweed genera Gelidium and Gracilaria, and consists of repeated agarobiose (L- and D-galactose) subunits2. During gelation, agarose polymers associate non-covalently and form a network of bundles whose pore sizes determine a gel&#39;s molecular sieving properties. The use of agarose gel electrophoresis revolutionized the separation of DNA. Prior to the adoption of agarose gels, DNA was primarily separated using sucrose density gradient centrifugation, which only provided an approximation of size. To separate DNA using agarose gel electrophoresis, the DNA is loaded into pre-cast wells in the gel and a current applied. The phosphate backbone of the DNA (and RNA) molecule is negatively charged, therefore when placed in an electric field, DNA fragments will migrate to the positively charged anode. Because DNA has a uniform mass/charge ratio, DNA molecules are separated by size within an agarose gel in a pattern such that the distance traveled is inversely proportional to the log of its molecular weight3. The leading model for DNA movement through an agarose gel is &#34;biased reptation&#34;, whereby the leading edge moves forward and pulls the rest of the molecule along4. The rate of migration of a DNA molecule through a gel is determined by the following: 1) size of DNA molecule; 2) agarose concentration; 3) DNA conformation5; 4) voltage applied, 5) presence of ethidium bromide, 6) type of agarose and 7) electrophoresis buffer. After separation, the DNA molecules can be visualized under uv light after staining with an appropriate dye. By following this protocol, students should be able to: 
 
1. Understand the mechanism by which DNA fragments are separated within a gel matrix 
2. Understand how conformation of the DNA molecule will determine its mobility through a gel matrix 
3. Identify an agarose solution of appropriate concentration for their needs 
4. Prepare an agarose gel for electrophoresis of DNA samples 
5. Set up the gel electrophoresis apparatus and power supply 
6. Select an appropriate voltage for the separation of DNA fragments 
7. Understand the mechanism by which ethidium bromide allows for the visualization of DNA bands 
8. Determine the sizes of separated DNA fragments 
&#160;

A basic protocol for the separation of DNA fragments using agarose gel electrophoresis is described.

Électrophorèse sur gel d&#39;agarose pour la séparation de fragments d&#39;ADN

Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from 100 bp to 25 kb1. Agarose is isolated from ...

Preparation of DNA-crosslinked Polyacrylamide Hydrogels

Mechanobiology is an emerging scientific area that addresses the critical role of physical cues in directing cell morphology and function. For example, the effect of tissue elasticity on cell function is a major area of mechanobiology research because tissue stiffness modulates with disease, development, and injury. Static tissue-mimicking materials, or materials that cannot alter stiffness once cells are plated, are predominately used to investigate the effects of tissue stiffness on cell functions. While information gathered from static studies is valuable, these studies are not indicative of the dynamic nature of the cellular microenvironment in vivo. To better address the effects of dynamic stiffness on cell function, we developed a DNA-crosslinked polyacrylamide hydrogel system (DNA gels). Unlike other dynamic substrates, DNA gels have the ability to decrease or increase in stiffness after fabrication without stimuli. DNA gels consist of DNA crosslinks that are polymerized into a polyacrylamide backbone. Adding and removing crosslinks via delivery of single-stranded DNA allows temporal, spatial, and reversible control of gel elasticity. We have shown in previous reports that dynamic modulation of DNA gel elasticity influences fibroblast and neuron behavior. In this report and video, we provide a schematic that describes the DNA gel crosslinking mechanisms and step-by-step instructions on the preparation DNA gels.

Our laboratory has developed DNA-crosslinked polyacrylamide hydrogels, a dynamic hydrogel system, to better understand the effects of modulating tissue stiffness on cell function. Here, we provide schematics, descriptions, and protocols to prepare these hydrogels.

Préparation de l&#39;ADN réticulé polyacrylamide hydrogels

Mechanobiology is an emerging scientific area that addresses the critical role of physical cues in directing cell morphology and function. For example, ...

Mucin Agarose Gel Electrophoresis: Western Blotting for High-molecular-weight Glycoproteins

Mucins, the heavily-glycosylated proteins lining mucosal surfaces, have evolved as a key component of innate defense by protecting the epithelium against invading pathogens. The main role of these macromolecules is to facilitate particle trapping and clearance while promoting lubrication of the mucosa. During protein synthesis, mucins undergo intense O-glycosylation and multimerization, which dramatically increase the mass and size of these molecules. These post-translational modifications are critical for the viscoelastic properties of mucus. As a result of the complex biochemical and biophysical nature of these molecules, working with mucins provides many challenges that cannot be overcome by conventional protein analysis methods. For instance, their high-molecular-weight prevents electrophoretic migration via regular polyacrylamide gels and their sticky nature causes adhesion to experimental tubing. However, investigating the role of mucins in health (e.g., maintaining mucosal integrity) and disease (e.g., hyperconcentration, mucostasis, cancer) has recently gained interest and mucins are being investigated as a therapeutic target. A better understanding of the production and function of mucin macromolecules may lead to novel pharmaceutical approaches, e.g., inhibitors of mucin granule exocytosis and/or mucolytic agents. Therefore, consistent and reliable protocols to investigate mucin biology are critical for scientific advancement. Here, we describe conventional methods to separate mucin macromolecules by electrophoresis using an agarose gel, transfer protein into nitrocellulose membrane, and detect signal with mucin-specific antibodies as well as infrared fluorescent gel reader. These techniques are widely applicable to determine mucin quantitation, multimerization and to test the effects of pharmacological compounds on mucins.

Mucins are high-molecular-weight glycoconjugates, with size ranging from 0.2 to 200 megadalton (MDa). As a result of their size, mucins do not penetrate conventional polyacrylamide gels and require larger pores for separation. We provide a detailed protocol for mucin agarose gel electrophoresis to assess relative quantification and study polymer assembly.

Mucine Agarose Gel Electrophoresis: Western Blot pour les glycoprotéines de haut poids moléculaire,


Mucins, the heavily-glycosylated proteins lining mucosal surfaces, have evolved as a key component of innate defense by protecting the epithelium against ...

Analysis of Mitochondrial Respiratory Chain Complexes in Cultured Human Cells using Blue Native Polyacrylamide Gel Electrophoresis and Immunoblotting

Mitochondrial respiration is performed by oxidative phosphorylation (OXPHOS) complexes within mitochondria. Internal and environmental factors can perturb the assembly and stability of OXPHOS complexes. This protocol describes the analysis of mitochondrial respiratory chain complexes by blue native polyacrylamide gel electrophoresis (BN-PAGE) in application to cultured human cells. First, mitochondria are extracted from the cells using digitonin, then using lauryl maltoside, the intact OXPHOS complexes are isolated from the mitochondrial membranes. The OXPHOS complexes are then resolved by gradient gel electrophoresis in the presence of the negatively charged dye, Coomassie blue, which prevents protein aggregation and ensures electrophoretic mobility of protein complexes towards the cathode. Finally, the OXPHOS complexes are detected by standard immunoblotting. Thus, BN-PAGE is a convenient and inexpensive technique that can be used to evaluate the assembly of entire OXPHOS complexes, in contrast to the basic SDS-PAGE allowing the study of only individual OXPHOS complex subunits.

This protocol demonstrates the analysis of mitochondrial respiratory chain complexes by blue native polyacrylamide gel electrophoresis. Here the method applied to cultured human cells is described.

Analyse des Complexes de chaîne respiratoire mitochondriale dans les cellules humaines cultivées à l’aide de bleu sur Native de Gel de Polyacrylamide et Immunoblotting

Mitochondrial respiration is performed by oxidative phosphorylation (OXPHOS) complexes within mitochondria. Internal and environmental factors can perturb ...

Detection of Glycosaminoglycans by Polyacrylamide Gel Electrophoresis and Silver Staining

Sulfated glycosaminoglycans (GAGs) such as heparan sulfate (HS) and chondroitin sulfate (CS) are ubiquitous in living organisms and play a critical role in a variety of basic biological structures and processes. As polymers, GAGs exist as a polydisperse mixture containing polysaccharide chains that can range from 4000 Da to well over 40,000 Da. Within these chains exists domains of sulfation, conferring a pattern of negative charge that facilitates interaction with positively charged residues of cognate protein ligands. Sulfated domains of GAGs must be of sufficient length to allow for these electrostatic interactions. To understand the function of GAGs in biological tissues, the investigator must be able to isolate, purify, and measure the size of GAGs. This report describes a practical and versatile polyacrylamide gel electrophoresis-based technique that can be leveraged to resolve relatively small differences in size between GAGs isolated from a variety of biological tissue types.

This report describes techniques to isolate and purify sulfated glycosaminoglycans (GAGs) from biological samples and a polyacrylamide gel electrophoresis approach to approximate their size. GAGs contribute to tissue structure and influence signaling processes via electrostatic interaction with proteins. GAG polymer length contributes to their binding affinity for cognate ligands.

Détection des glycosaminoglycanes par électrophorèse sur gel de polyacrylamide et coloration à l’argent

Sulfated glycosaminoglycans (GAGs) such as heparan sulfate (HS) and chondroitin sulfate (CS) are ubiquitous in living organisms and play a critical role ...

Dissecting, Fixing, and Visualizing the Drosophila Pupal Notum

The pupae of Drosophila melanogaster are immobile for several days during metamorphosis, during which they develop a new body with a thin transparent adult integument. Their immobility and transparency make them ideal for in vivo live imaging experiments. Many studies have focused on the dorsal epithelial monolayer of the pupal&#160;notum because of its accessibility and relatively large size. In addition to the studies of epithelial mechanics and development, the notum has been an ideal tissue to study wound healing. After an injury, the entire epithelial repair process can be captured by live imaging over 6-12 h. Despite the popularity of the notum for live imaging, very few published studies have utilized fixed notum samples. Fixation and staining are common approaches for nearly all other Drosophila tissues, taking advantage of the large repertoire of simple cellular stains and antibodies. However, the pupal notum is fragile and prone to curling and distortion after removal from the body, making it challenging to complement live imaging. This protocol offers a straightforward method for fixing and staining the pupal notum, both intact and after laser-wounding. With this technique, the ventral side of the pupa is glued down to a coverslip to immobilize the pupa, and the notum is carefully removed, fixed, and stained. The notum epithelium is mounted on a slide or between two coverslips to facilitate imaging from the tissue&#39;s dorsal or ventral side.

Dissecting, Fixing, and Visualizing the Drosophila Pupal Notum

The present protocol details the preparation and visualization of fixed tissue of the Drosophila pupal notum. It can be used for either intact or wounded tissue, and the original architecture of the tissue is preserved. The procedures for dissecting, fixing, and staining are all described in this article.

Disséquer, fixer et visualiser la nymphe de la drosophile Notum

The pupae of Drosophila melanogaster are immobile for several days during metamorphosis, during which they develop a new body with a thin transparent ...