Les auteurs tiennent à remercier l&#39;Institut médical Howard Hughes pour une science UF pour l&#39;attribution de la vie à AH

1. Préparation des cassettes <ol><li> Avant et arrière Clean plaques d&#39;un employé Invitrogen NuPAGE cassette de Novex minigel, ou d&#39;autres cassettes minigel commerciale. </li><li> Préparer ml environ 1-3 de plastique époxy et bien mélanger selon les instructions du fabricant. </li><li> Appliquer la résine époxy à l&#39;arête intérieure de la plaque avant, où les deux plaques ne viennent en contact lorsque la cassette est assemblé. </li><li> Coulisser un morceau de papier filtre ou de carton à travers la fente inférieure de la plaque arrière lors de l&#39;assemblage de la cassette pour éviter l&#39;excès de résine époxy sceller l&#39;ouverture. Retirez le papier du filtre dès que les deux plaques sont assemblées. </li><li> Placez clips liant le long des bords pour un joint étanche, et laissez-les reposer toute la nuit cassettes pour l&#39;époxy pour durcir complètement. </li><li> Sceller la fente sur le bas de la plaque arrière avec du ruban électrique. </li></ol> 2. Coulage du gel de résolution <ol><li> Pour un flacon de 125 ml côté du bras, ajouter ce qui suit:2,25 ml de 4X Tris-Base gel de résolution tampon pH 8,70, 3,50 ml d&#39;acrylamide 30,8% / bisacrylamide, 3,25 ml de type I eau de qualité réactif (IRG-H 2 O) pour un volume total de 9 ml. </li><li> Dégazer la solution pendant 10 min en attachant le bras latéral du récipient à un appareil à vide, et placer un bouchon inversé sur le dessus de la fiole. </li><li> Transférer le 9 ml de solution dégazée à un 50 ml polypropylène centrifugeuse tube jetable et ajouter 30 ul de persulfate ammounium 10% (APS) et 6 ul de N, N, N &#39;, N&#39;,-tétraméthyléthylènediamine (TEMED) et mélanger ainsi en agitant la solution pour éviter l&#39;introduction de bulles. </li><li> Verser immédiatement la solution dans la cassette, ce qui lui permet de fonctionner sur le côté de la cassette pour éviter toute formation de bulles entre les plaques de gel. Remplissez la cassette à environ 2 cm du bord supérieur de l&#39;avant (plus courte) plaque. </li><li> Doucement, ajoutez 0,5 ml de 1-butanol saturé d&#39;un tampon 1X gel de résolution et de laisser le gel polymériser pour 1heures. </li></ol> 3. Verser le gel d&#39;empilement <ol><li> Ajoutez le code suivant dans une fiole de 125 ml côté du bras: 0,75 ml de 4X Tris-Base pH du gel de tampon d&#39;empilage 6,80, 0,40 ml d&#39;acrylamide 30,8% / bisacrylamide, 1,85 ml de l&#39;IRG-H 2 O pour un volume total de 3 ml. </li><li> De-gaz de la solution pendant 10 min comme décrit dans l&#39;étape 2. </li><li> Alors que la solution de gel d&#39;empilement est de dégazage, verser le 1-butanol saturé d&#39;un tampon 1X gel de résolution à partir du haut du gel de résolution. Rincez la partie supérieure du gel résoudre une fois avec du GRI-H 2 O. Verser l &#39;eau et utiliser un papier absorbant ou filtre Whatman pour absorber tout le liquide restant. </li><li> Retirer 3 ml de votre solution dégazée et ajouter 9 pi de l&#39;APS 10% et 2 pi de TEMED et bien mélanger. </li><li> Verser rapidement la solution dans la cassette, ce qui lui permet de fonctionner sur le côté de la cassette jusqu&#39;à ce qu&#39;il se trouve à proximité du bord supérieur de la plaque de gel plus courte devant. </li><li> Insérer un peigne gel propre dans la partie supérieure de la cassette de gel,placez un clip liant sur la cassette sur le peigne pour le maintenir en place. Laissez le gel d&#39;empilement polymériser pendant 1 heure ou toute la nuit. </li></ol> 4. Stockage du gel <ol><li> Lorsque le gel est complètement polymérisée, retirer les clips de liant et placer la cassette dans un sachet en plastique thermosoudable, ou un sac en plastique refermable. </li><li> Verser 20 ml de tampon 1X gel d&#39;empilement qui contient de l&#39;azide de sodium à 0,1% dans la poche. </li><li> Chauffer sceller le sachet pendant 5 secondes et assurez-vous qu&#39;il n&#39;ya aucune fuite dans le sac. </li><li> Conserver le gel à 4 ° C. </li></ol> 5. Préparation des échantillons <ol><li> Dans un microtube à paroi mince, ajouter jusqu&#39;à 6,5 ul de l&#39;échantillon de protéines, 7,5 ul de 2x Novex Tris-Glycine tampon d&#39;échantillon SDS, 1 pl de 100 mM (DTT) et dithiothréitol GRI-H 2 O (si nécessaire) pour un volume total de 15 pi. </li><li> Mélanger l&#39;échantillon par vortex, et brièvement centrifuger l&#39;échantillon pour amener le contenu de la partie inférieure du tube. </li> &lt;p&gt; Dénaturer l&#39;échantillon à 70 ° C pendant 10 min. </li></ol> 6. Exécution du gel <ol><li> Mettre en place l&#39;appareil à gel Novex. </li><li> Ajouter 200 ml de tampon de migration Tris-Glycine qui contient 500 ul d&#39;antioxydant NuPAGE à la chambre intérieure, et 300 ml de Tris-Glycine Running tampon à la chambre extérieure. </li><li> Chargez les échantillons de protéines dénaturées (15 pi) dans les puits en utilisant un gel de chargement pointe de pipette. </li><li> Exécutez le gel à 200 V pour 60-70 min. </li></ol> 7. La coloration du gel <ol><li> Retirer la cassette de l&#39;appareil de gel et rompre le joint époxy en faisant levier les deux plaques de gel de plastique en dehors aide d&#39;un couteau de gel ou d&#39;une spatule rigide. Soigneusement séparer le gel des plaques, en prenant soin de ne pas déchirer le gel. </li><li> Rincer le gel avec du GRI-H 2 O trois fois avec une légère agitation. </li><li> Colorer le gel avec Invitrogen SimplyBlue SafeStain pendant 1 heure sous agitation douce. </li><li> Décanter la solution de coloration et dedécolorer le gel avec du GRI-H 2 O. </li></ol> 8. Nettoyage des cassettes <ol><li> Utilisez une spatule en métal pour gratter ou peler tout l&#39;époxy sur les cassettes afin qu&#39;ils puissent être réutilisés à nouveau. </li><li> Laver chacun des plaques de gel de plastique avec une solution de détergent doux, et rincez bien avec de l&#39;eau du robinet puis à l&#39;eau distillée. </li></ol> 9. Les résultats représentatifs Parce époxy formera un joint étanche à l&#39;eau, (Figure 1), les cassettes ne fuira pas quand la solution d&#39;acrylamide est versé dans les. En outre, les cassettes de gel peut être facilement ouverte pour récupérer le gel pour la coloration, et la résine époxy peut être enlevée des cassettes pour permettre leur maintien réutilisation. Comme le montre la figure 2, le gel montre une séparation claire des marqueurs protéiques et des bandes d&#39;échantillons appropriés pour l&#39;électrophorèse des protéines de routine. <img src="/files/ftp_upload/3465/3465fig1.jpg" alt="Figure 1" /> Figure 1. Applications d&#39;époxy pour sceller des cassettes MiniGel. une couche mince de résine époxy est appliquée à l&#39;arête de l&#39;une des cassettes avant le montage. Afin de souligner l&#39;époxy, il était faux de couleur rouge sur cette image. <img src="/files/ftp_upload/3465/3465fig2.jpg" alt="Figure 2" /> Figure 2. Séparation des protéines sur un gel SDS-Tris-glycine versait à l&#39;aide réutilisés cassettes MiniGel. SDS-PAGE a été réalisée comme décrit ci-dessus. Après électrophorèse du gel a été retiré de la cassette minigel, teinté en SimplyBlue SafeStain selon les instructions du fabricant, et photographiées à l&#39;aide de la lumière blanche transmise. Lane M contient SeeBlue normes Plus2 protéines pré-teint. Les masses moléculaires des protéines dans piste M sont indiqués sur le côté droit du gel en kilodaltons (kDa). Les autres voies montrent protéines à partir d&#39;un lysat clarifié à partir de bactéries exprimant une glutathion-S-transférase (GST)-protéine marquée. Les protéines indiquées sont celles lavé de la gperles lutathione avant élution de la protéine GST-étiquetés.

<ol>
	<li>Laemmli, U. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cleavage+of+structural+proteins+during+the+assembly+of+the+head+of+bacteriophage+T4.">Cleavage of structural proteins during the assembly of the head of bacteriophage T4.</a> Nature. 227, 680-685 (1970).</li><li>Raymond, S., Weintraub, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acrylamide+gel+as+a+supporting+medium+for+zone+electrophoresis.">Acrylamide gel as a supporting medium for zone electrophoresis.</a> Science. 130, 711-711 (1959).</li><li>Sch&#228;gger, H., von Jagow, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tricine-sodium+dodecyl+sulfate-polyacrylamide+gel+electrophoresis+for+the+separation+of+proteins+in+the+range+from+1+to+100+kDa.">Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa.</a> Anal. Biochem. 166, 368-379 (1987).</li><li>Shapiro, A. L., Vi&#241;uela, E., Maizel, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+weight+estimation+of+polypeptide+chains+by+electrophoresis+in+SDS-polyacrylamide+gels.">Molecular weight estimation of polypeptide chains by electrophoresis in SDS-polyacrylamide gels.</a> Biochem. Biophys. Res. Commun. 28, 815-820 (1967).</li></ol>

Pas de conflits d&#39;intérêt déclarés.

Électrophorèse sur gel des protéines est une méthode indispensable pour les laboratoires de biologie moléculaire les plus. Minigels disponibles dans le commerce de fournir un niveau élevé de confort, mais peuvent être coûteux. Ici, nous montrons comment réutiliser les cassettes MiniGel à partir d&#39;une marque populaire de minigels disponibles dans le commerce. Cette méthode conserve la commodité d&#39;avoir une source prête à l&#39;emploi de gels, mais à une fraction du coût de minigels disponibles da...

<table border="1"><tbody><tr><td> Nom de réactif </td><td> Entreprise </td><td> Numéro de catalogue </td><td> Commentaires </td></tr><tr><td> Invitrogen NuPAGE Novex minigel cassette (1,0 mm) </td><td> Invitrogen </td><td> NC2010 </td><td> Disponible en 2, 5, 10, 12, 15 et 2D puits </td></tr><tr><td> Epoxy plastique </td><td> Loctite </td><td> 108771 </td><td></td></tr><tr><td> Ruban électrique </td><td> Scotch </td><td></td><td></td></tr><tr><td> 125 ml flacon bras latéral </td><td> Pyrex </td><td> 5360 </td><td></td></tr><tr><td> 4X Tris-Base gel de résolution du tampon </td><td></td><td></td><td> 1,5 M Tris-base pH 8,7 </td></tr><tr><td> acrylamide </td><td> Fisher Scientific </td><td> 01065-500 </td><td></td></tr><tr><td> Bis-acrylamide </td><td> MP Biomedicals, Inc </td><td> 800173 </td><td></td></tr><tr><td> Solution d&#39;acrylamide 30,8% </td><td></td><td></td><td> 30:0.8 acrylamide: bis-acrylamide dans les GRI-H 2 O </td></tr><tr><td> APS 10% </td><td> Fisher Scientific </td><td> BP179-25 </td><td> 0,1 g de persulfate d&#39;ammonium dans 1 ml O GRI-H 2, préparée immédiatement avant utilisation </td></tr><tr><td> TEMED </td><td> Organics Arcos </td><td> 138450500 </td><td></td></tr><tr><td> 1-butanol saturé avec un tampon 1X gel de résolution </td><td></td><td></td><td> 05:02 1-butanol à 1X gel de résolution du tampon </td></tr><tr><td> 4X Tris-Base tampon de gel d&#39;empilement </td><td></td><td></td><td> 1,5 M Tris-base pH 6,8 </td></tr><tr><td> Gel peigne </td><td> Invitrogen </td><td> NC3015 </td><td> 15 bien, 1,0 mm d&#39;épaisseur </td></tr><tr><td> Scellant Impulse </td><td> Equipement TEW Chauffage électrique </td><td></td><td></td></tr><tr><td> Pochette de chaleur plombable </td><td> Kapak / Scotchpak </td><td></td><td> 1 taille pinte (6,5 &quot;x 8&quot;) </td></tr><tr><td> Tampon de stockage Gel </td><td></td><td></td><td> 1X tampon de gel d&#39;empilement avec de l&#39;azoture de sodium à 0,1% </td></tr><tr><td> Novex SDS tampon d&#39;échantillon Tris-Glycine (2X) </td><td> Invitrogen </td><td> LC2676 </td><td></td></tr><tr><td> TNT </td><td> FisherBiotech </td><td> BP172-5 </td><td></td></tr><tr><td> 10X tampon de migration Tris-Gylcine </td><td> Invitrogen </td><td> LC2675 </td><td> 1L de bouillon 10X: 29,0 g de Tris-Base, 144,0 g de glycine, 10,0 g de SDS </td></tr><tr><td> XCell SureLock Mini-Cell </td><td> Invitrogen </td><td> EI0001 </td><td></td></tr><tr><td> Couteau Gel </td><td> Invitrogen </td><td> EI9010 </td><td></td></tr><tr><td> SimplyBlue Safe Stain </td><td> Invitrogen </td><td> LC6065 </td&gt; <td></td></tr></tbody></table>

pouring and running a protein gel by reusing commercial cassettes

L&#39;évaluation des protéines à l&#39;aide de sodium dodécyl polyacrylamide gel de sulfate de l&#39;électrophorèse (SDS-PAGE) l&#39;analyse est une technique couramment utilisée par les chercheurs en biologie moléculaire et de biochimie 1-4. Pour les laboratoires qui effectuent des analyses quotidiennes de protéines, le coût de gels de polyacrylamide disponibles dans le commerce (~ $ 10/gel) peuvent être considérables au fil du temps. Pour atténuer ce coût, certains chercheurs à préparer leurs propres gels de polyacrylamide. Les méthodes traditionnelles de verser ces gels utilisent généralement l&#39;équipement spécialisé et des plaques de gel de verre qui peuvent être coûteux et s&#39;oppose versant gels de nombreuses et de les stocker pour une utilisation future. En outre, la manipulation de plaques de verre pendant le nettoyage ou le gel versant peut se traduire par un bris accidentel de créer un danger pour la sécurité, ce qui peut empêcher leur utilisation dans les classes de premier cycle en laboratoire. Notre protocole montre comment verser gels de protéines multiples simultanément par recyclage Invitrogen cassettes NuPAGE MiniGel Novex, et peu coûteux mariaux achetés à un magasin de rénovation domiciliaire. Cette méthode économique et rationalisé comprend un moyen de stocker les gels à 4 ° C pendant quelques semaines. En réutilisant les cassettes de gel en plastique à partir de gels disponibles dans le commerce, les laboratoires qui exécutent des gels de protéine fréquents peuvent réduire les coûts importants et aider l&#39;environnement. En outre, cassettes de gel en plastique sont extrêmement résistants à la rupture, ce qui les rend idéal pour les salles de classe en laboratoire de premier cycle.

Watch this Scientific Journal Video about Pouring and Running a Protein Gel by reusing Commercial Cassettes at JoVE.com

Pouring and Running a Protein Gel by reusing Commercial Cassettes

Notre protocole montre comment verser gels de protéines multiples à la fois par le recyclage Invitrogen cassettes NuPAGE MiniGel Novex, et des matériaux peu coûteux achetés dans un magasin de rénovation domiciliaire. Cette méthode économique et rationalisé comprend un moyen de stocker les gels à 4 ° C pendant quelques semaines. En réutilisant les cassettes de gel en plastique à partir de gels disponibles dans le commerce, les laboratoires qui exécutent des gels de protéine fréquents peuvent réduire les coûts importants et aider l&#39;environnement.

Verser et exécution d&#39;un gel de protéine en réutilisant Cassettes commerciaux

The evaluation of proteins using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis is a common technique used by biochemistry ...

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24.8K vues.  University of Florida. Notre protocole montre comment verser gels de protéines multiples à la fois par le recyclage Invitrogen cassettes NuPAGE MiniGel Novex, et des matériaux peu coûteux achetés dans un magasin de rénovation domiciliaire. Cette méthode économique et rationalisé comprend un moyen de stocker les gels à 4 ° C pendant quelques semaines. En réutilisant les cassettes de gel en plastique à partir de gels disponibles dans le commerce, les laboratoires qui exécutent des g...

Vidéo: Pouring and Running a Protein Gel by reusing Commercial Cassettes

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 ...

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Biologie

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

Coloration des protéines dans les gels de Coomassie G-250, sans solvant et l&#39;acide acétique organique

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

Utiliser le système de fractionnement GELFREE 8100 pour les Poids Moléculaire à base de fractionnement avec récupération de la phase liquide

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag

Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a demand for general methods that do not require target protein specific optimization1 . To achieve this, purification tags that genetically can be fused to the gene of interest are commonly used2 . The most widely used affinity handle is the hexa-histidine tag, which is suitable for purification under both native and denaturing conditions3 . The metabolic burden for producing the tag is low, but it does not provide as high specificity as competing affinity chromatography based strategies1,2.
Here, a bispecific purification tag with two different binding sites on a 46 amino acid, small protein domain has been developed. The albumin-binding domain is derived from Streptococcal protein G and has a strong inherent affinity to human serum albumin (HSA). Eleven surface-exposed amino acids, not involved in albumin-binding4 , were genetically randomized to produce a combinatorial library. The protein library with the novel randomly arranged binding surface (Figure 1) was expressed on phage particles to facilitate selection of binders by phage display technology. Through several rounds of biopanning against a dimeric Z-domain derived from Staphylococcal protein A5, a small, bispecific molecule with affinity for both HSA and the novel target was identified6 .
The novel protein domain, referred to as ABDz1, was evaluated as a purification tag for a selection of target proteins with different molecular weight, solubility and isoelectric point. Three target proteins were expressed in Escherishia coli with the novel tag fused to their N-termini and thereafter affinity purified. Initial purification on either a column with immobilized HSA or Z-domain resulted in relatively pure products. Two-step affinity purification with the bispecific tag resulted in substantial improvement of protein purity. Chromatographic media with the Z-domain immobilized, for example MabSelect SuRe, are readily available for purification of antibodies and HSA can easily be chemically coupled to media to provide the second matrix.
This method is especially advantageous when there is a high demand on purity of the recovered target protein. The bifunctionality of the tag allows two different chromatographic steps to be used while the metabolic burden on the expression host is limited due to the small size of the tag. It provides a competitive alternative to so called combinatorial tagging where multiple tags are used in combination1,7.

A novel and highly efficient two-step affinity chromatography protocol has been developed and is described in detail. The method is based on a small purification tag with two inherent affinities and is applicable to a wide range of target proteins with different properties.

Protein Purification Orthogonal Animé par une étiquette d&#39;affinité bispécifiques petits

Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a ...

Improving the Success Rate of Protein Crystallization by Random Microseed Matrix Screening

Random microseed matrix screening (rMMS) is a protein crystallization technique in which seed crystals are added to random screens. By increasing the likelihood that crystals will grow in the metastable zone of a protein's phase diagram, extra crystallization leads are often obtained, the quality of crystals produced may be increased, and a good supply of crystals for data collection and soaking experiments is provided. Here we describe a general method for rMMS that may be applied to either sitting drop or hanging drop vapor diffusion experiments, established either by hand or using liquid handling robotics, in 96-well or 24-well tray format.

Here we describe a general method for random microseed matrix screening. This technique is shown to significantly increase the success rate of protein crystallization screening experiments, reduce the need for optimization, and provide a reliable supply of crystals for data collection and ligand-soaking experiments.

Améliorer le taux de réussite de cristallisation de protéines par hasard Microseed Matrice dépistage

Random microseed matrix screening  (rMMS) is a protein crystallization technique in which seed crystals are added  to random screens. By increasing the ...

Protein-protein Interactions Visualized by Bimolecular Fluorescence Complementation in Tobacco Protoplasts and Leaves

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular fluorescence complementation (BiFC) is an in vivo method to monitor such interactions in plant cells. In the presented protocol the investigated candidate proteins are fused to complementary halves of fluorescent proteins and the respective constructs are introduced into plant cells via agrobacterium-mediated transformation. Subsequently, the proteins are transiently expressed in tobacco leaves and the restored fluorescent signals can be detected with a confocal laser scanning microscope in the intact cells. This allows not only visualization of the interaction itself, but also the subcellular localization of the protein complexes can be determined. For this purpose, marker genes containing a fluorescent tag can be coexpressed along with the BiFC constructs, thus visualizing cellular structures such as the endoplasmic reticulum, mitochondria, the Golgi apparatus or the plasma membrane. The fluorescent signal can be monitored either directly in epidermal leaf cells or in single protoplasts, which can be easily isolated from the transformed tobacco leaves. BiFC is ideally suited to study protein-protein interactions in their natural surroundings within the living cell. However, it has to be considered that the expression has to be driven by strong promoters and that the interaction partners are modified due to fusion of the relatively large fluorescence tags, which might interfere with the interaction mechanism. Nevertheless, BiFC is an excellent complementary approach to other commonly applied methods investigating protein-protein interactions, such as coimmunoprecipitation, in vitro pull-down assays or yeast-two-hybrid experiments.

Formation of protein complexes in vivo can be visualized by bimolecular fluorescence complementation. Interaction partners are fused to complementary parts of fluorescent tags and transiently expressed in tobacco leaves, resulting in a reconstituted fluorescent signal upon close proximity of the two proteins.

Interactions protéine-protéine visualisés par fluorescence bimoléculaire complémentation en protoplastes de tabac et des feuilles

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

Fecal glucocorticoïdes Analyse: Surveillance surrénale non invasive en Equidés

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

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 ...

A Simple and Inexpensive Running Wheel Model for Progressive Resistance Training in Mice

Previously developed rodent resistance-based exercise models, including synergistic ablation, electrical stimulation, weighted-ladder climbing, and most recently, weighted-sled pulling, are highly effective at providing a hypertrophic stimulus to induce skeletal muscle adaptations. While these models have proven invaluable for skeletal muscle research, they are either invasive or involuntary and labor-intensive. Fortunately, many rodent strains voluntarily run long distances when given access to a running wheel. Loaded wheel running (LWR) models in rodents are capable of inducing adaptations commonly observed with resistance training in humans, such as increased muscle mass and fiber hypertrophy, as well as stimulation of muscle protein synthesis. However, the addition of moderate wheel load either fails to deter mice from running great distances, which is more reflective of an endurance/resistance training model, or the mice discontinue running nearly entirely due to the method of load application. Therefore, a novel high-load wheel running model (HLWR) has been developed for mice where external resistance is applied and progressively increased, enabling mice to continue running with much higher loads than previously utilized. Preliminary results from this novel HLWR model suggest it provides sufficient stimulus to induce hypertrophic adaptations over the 9 week training protocol. Herein, the specific procedures to execute this simple yet inexpensive progressive resistance-based exercise training model in mice are described.

This procedure describes a translatable progressive loaded running wheel resistance training model in mice. The primary advantage of this resistance training model is that it is entirely voluntary, thus reducing stress for the animals and the burden on the researcher.

Un modèle de roue de roulement simple et peu coûteux pour l’entraînement progressif en résistance chez la souris

Previously developed rodent resistance-based exercise models, including synergistic ablation, electrical stimulation, weighted-ladder climbing, and most ...

Simplification des études d’angiogenèse avec un dosage à matrice modifiée

Modified In Vivo Matrix Gel Plug Assay for Angiogenesis Studies

Several models have been developed to investigate angiogenesis in vivo. However, most of these models are complex and expensive, require specialized equipment, or are hard to perform for subsequent quantitative analysis. Here we present a modified matrix gel plug assay to evaluate angiogenesis in vivo. In this protocol, vascular cells were mixed with matrix gel in the presence or absence of pro-angiogenic or anti-angiogenic reagents, and then subcutaneously injected into the back of recipient mice. After 7 days, phosphate buffer saline containing dextran-FITC is injected via the tail vein and circulated in vessels for 30 min. Matrix gel plugs are collected and embedded with tissue embedding gel, then 12 &#181;m sections are cut for fluorescence detection without staining. In this assay, dextran-FITC with high molecular weight (~150,000 Da) can be used to indicate functional vessels for detecting their length, while dextran-FITC with low molecular weight (~4,400 Da) can be used to indicate the permeability of neo-vessels. In conclusion, this protocol can provide a reliable and convenient method for the quantitative study of angiogenesis in vivo.

Pleins feux sur l’auteur : Étude de l’angiogenèse et de la perméabilité des vaisseaux à l’aide d’un dosage du bouchon de gel matriciel modifié

The method presented here can evaluate the effect of reagents on angiogenesis or vascular permeability in vivo without staining. The method uses dextran-FITC injection via the tail vein to visualize neo-vessels or vascular leakage.

Essai de bouchon de gel matriciel in vivo modifié pour les études d’angiogenèse

Several models have been developed to investigate angiogenesis in vivo. However, most of these models are complex and expensive, require specialized ...