Nous reconnaissons avec gratitude le soutien technique de Sini Miettinen, Minna Pöllänen et Taru Rautavesi. Nous remercions Helka Nurkkala pour fournir des cellules HEK 293 exprimant double-Strep-GFP étiqueté et Pekka Evijärvi pour la fourniture de matériel d&#39;enregistrement sonore. Ce travail a été financé par l&#39;Académie de Finlande, accorder les numéros 138329, 134684 et 258978.

1. Réticulation de Strep-Tactin polyméthacrylate résine avec Bis (sulfosuccinimidyl) subérate (BS3) <ol><li> Équilibrer un microtube scellé contenant 2 mg de BS3 agent de réticulation à température ambiante. ATTENTION: BS3 est une substance dangereuse. Portez des gants et des lunettes de protection. </li><li> Résine de polyméthacrylate Remettre en suspension Strep-Tactin (suspension à 50% dans 100 mM Tris-HCl, pH 8,0; EDTA 1 mM, NaCl 150 mM) par une brève agitation vigoureuse et à transférer immédiatement 600 ul de la suspension à une colonne de centrifugation à l&#39;aide d&#39;un embout de pipette avec la fin coupée. </li><li> Centrifuger à 1500 g pendant 30 secondes à la température ambiante. Jeter l&#39;effluent et ajouter 450 ul de tampon phosphate salin (PBS) à la colonne. </li><li> Répétez l&#39;étape précédente 2 fois plus de remplacer complètement le tampon Tris avec du PBS et laisser la résine dans 430 pi de PBS ajusté à pH 8,0 après la dernière centrifugation. </li><li> Piquer la feuille du microtube avec BS3 avec une pointe de pipette contenant 1001, l d&#39;eau ultra-pure. Dissoudre la poudre dans de l&#39;eau BS3 en pipettant doucement vers le haut et vers le bas et ajouter immédiatement 20 pi de la solution de la colonne de centrifugation. La concentration finale de BS3 dans la réaction de réticulation est d&#39;environ 1,2 mM. </li><li> Tournez la colonne pendant 30 min à température ambiante. Vérifier que la résine est mélangée convenablement avec la solution de BS3. </li><li> Pour stopper la réaction, ajouter 6 ul de Tris-HCl 3M, pH 7,5 et de faire tourner la colonne pendant 15 minutes supplémentaires à température ambiante. </li><li> Centrifuger à 1500 g pendant 30 secondes à la température ambiante. Jeter l&#39;effluent à travers et remettre en suspension la résine réticulée dans 450 pi de Tris solution saline tamponnée avec du Tween 20 (TBST). Répétez la centrifugation et les étapes de lavage deux fois de plus. Lors de la dernière étape, remettre en suspension la résine dans 450 pi de TBS. </li><li> Transférer la suspension de résine dans un nouveau tube à l&#39;aide d&#39;une pipette avec l&#39;extrémité coupée. Reprendre la résine dans la colonne de gauche avec un autre 450 pi de TBS et transférer dans le même tube. Répétez ee dernière étape une fois de plus pour assurer un transfert maximal de la résine de la colonne sur le tube. </li><li> Laissez le tube reposer pendant 10 min à température ambiante et ajuster le volume à 600 pi en enlevant l&#39;excès SCT. La résine est prêt pour une utilisation immédiate (recommandé) ou peut être conservé à 4 ° C, sans congélation. </li></ol> 2. Liaison de la protéine appât Twin-Strep-marqués et associés Complexes à la Strep-Tactin polyméthacrylate résine réticulée <ol><li> Centrifugeuse de 1 ml de l&#39;échantillon de protéine d&#39;appât dans le tampon de liaison à 17 000 xg pendant 10 min à 4 ° C et transférer le surnageant dans un tube frais. </li><li> Pour minimiser la liaison de protéines endogènes biotinylés à la résine Strep-Tactin, ajouter de l&#39;avidine à une concentration finale de 100 pg / ml et de tourner pendant 15 min à 4 ° C. <ol><li> Si la résine réticulée de l&#39;étape 1.10 a été stocké pendant une longue période de temps à 4 ° C, de recueillir la résine par centrifugation à 400 xg for 1 min à 4 ° C, éliminer le surnageant et laver la résine avec 1 ml de TBST. Répétez la centrifugation et lavage étapes deux fois, d&#39;abord avec TBST puis avec le SCT. Centrifuger le tube à 400 x g pendant 1 min à 4 ° C et ajuster le volume d&#39;origine en enlevant l&#39;excès de TBS. </li></ol></li><li> Remettre en suspension la résine Strep-Tactin réticulé au vortex. Ajouter immédiatement 50 pi de la suspension de résine dans le tube contenant l&#39;échantillon de protéine appât en utilisant un embout de pipette de coupe et faire tourner pendant encore 30 min à 4 ° C. </li><li> En attendant, mettre thermomixer à 55 ° C et préchauffer à 500 pi de tampon d&#39;élution pour une utilisation dans l&#39;étape 3.1. </li><li> Centrifuger à 400 g pendant 1 min à 4 ° C. Jeter le surnageant et laver la résine sur un agitateur pendant 5 min à 4 ° C avec 1 ml de la pré-réfrigérés tampon de lavage n ° 1. Répétez la centrifugation et les étapes de lavage trois fois. Lors de la dernière étape, remettre en suspension la résine dans 250 ul de tampon de lavage n ° 2. </li><li> Tr<html> ansfer la suspension de résine d&#39;une colonne de centrifugation douce. Remettre en suspension la résine restant dans le tube dans un autre 250 ul de tampon de lavage n ° 2 et le transférer dans la même colonne. </li><li> Centrifugeuse à 400 g pendant 3 min à 4 ° C, jeter le accréditives et transférer la colonne à un dauphin nez 2 nouveau tube de ml. Procéder immédiatement à l&#39;étape d&#39;élution ci-dessous. </li></ol> 3. L&#39;élution de la protéine spécifique Complexes <ol><li> Ajouter 150 pi de tampon d&#39;élution préchauffé provenant de l&#39;étape 2.4 de la colonne de centrifugation. </li><li> Incuber dans thermomixer pendant 5 min à 55 ° C, en agitant à 1400 tours par minute. </li><li> Centrifuger à 1500 g pendant 1 min à température ambiante. </li><li> Jeter la colonne et stocker les protéines cibles purifiés à ≤ -20 ° C. </li></ol><table border="0" cellpadding="0" cellspacing="0" width="598"><tbody><tr height="20"><td colspan="2" height="20" style="height:20px;width:597px;"> Tampon phosphate salin (PBS) </td></tr>0 &quot;&gt; <td height="20" style="height:20px;width:299px;"> Na 2 HPO 4 </td><td style="width:299px;"> 10 mM </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> KH 2 PO 4 </td><td style="width:299px;"> 2 mM </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> NaCl </td><td style="width:299px;"> 137 mM </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> KCl </td><td style="width:299px;"> 2,7 mM </td></tr><tr height="20"><td colspan="2" height="20" style="height:20px;width:597px;"> pH ajusté à 7,4, sauf indication contraire (pH 8,0 à la section 1.4) </td></tr><tr height="20"><td colspan="2" height="20" style="height:20px;width:597px;"> Tris solution saline tamponnée (SCT) </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> Tris-HCl, pH 7,4 </td><td style="width:299px;"> 50 mM </td></tr><tr height="20"><td hei<html> ght = le style &quot;20&quot; = &quot;height: 20px; largeur: 299px;&quot;&gt; NaCl </td><td style="width:299px;"> 150 mM </td></tr><tr height="20"><td colspan="2" height="20" style="height:20px;width:597px;"> Tris solution saline tamponnée avec du Tween 20 (TBST) </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> Tris-HCl, pH 7,4 </td><td style="width:299px;"> 50 mM </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> NaCl </td><td style="width:299px;"> 150 mM </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> Tween 20 </td><td style="width:299px;"> 0,1% (v / v) </td></tr><tr height="20"><td colspan="2" height="20" style="height:20px;width:597px;"> Tampon de liaison </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> Tris-HCl, pH 8,0 </td><td style="width:299px;"> 25 mM </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> NaCl </td>x &quot;&gt; 550 mM </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> NaF </td><td style="width:299px;"> 5 mM </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> EDTA </td><td style="width:299px;"> 0,5 mM </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> Glycérol </td><td style="width:299px;"> 10% (v / v) </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> PMSF </td><td style="width:299px;"> 0,1 mM </td></tr><tr height="20"><td colspan="2" height="20" style="height:20px;width:597px;"> Laver tampon # 1 </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> Tris-HCl, pH 8,0 </td><td style="width:299px;"> 25 mM </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> NaCl </td><td style="width:299px;"> 500 mM </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> NaF </<html> td&gt; <td style="width:299px;"> 5 mM </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> EDTA </td><td style="width:299px;"> 0,4 mM </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> Igepal CA-630 </td><td style="width:299px;"> 0,2% (v / v) </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> Glycérol </td><td style="width:299px;"> 5% (v / v) </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> PMSF </td><td style="width:299px;"> 0,1 mM </td></tr><tr height="20"><td colspan="2" height="20" style="height:20px;width:597px;"> Laver tampon # 2 </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> Tris-HCl, pH 8,0 </td><td style="width:299px;"> 25 mM </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> NaCl </td><td style="width:299px;"> 150 mM </td></tr><tr height="20">2 &quot;height =&quot; &quot;style =&quot; 20 hauteur: 20px; largeur: 597px; &quot;&gt; tampon d&#39;élution </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> Tris-HCl, pH 8,0 </td><td style="width:299px;"> 25 mM </td></tr><tr height="22"><td height="22" style="height:22px;width:299px;"> SDS </td><td style="width:299px;"> 1% (p / v) </td></tr></tbody></table> Tableau 1. Tampons utilisés dans la présente étude.

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Aucun conflit d&#39;intérêt déclaré.

Le protocole ci-dessus peut être utilisé pour purifier n&#39;importe quelle protéine appât double-Strep-marqué d&#39;intérêt et ses complexes sont associés à n&#39;importe quel tampon convenable qui ne contient pas de la biotine ou des dénaturants forts. Dans la version actuelle du protocole, de liaison et de lavage sont effectuées dans des conditions relativement strictes en la présence de forte teneur en sel et un détergent non-ionique. Bien que cela se traduit par moins de fond, des complexes de protéin...

Au cours des dernières années, la technologie Strep-tag a été largement utilisé dans de nombreux domaines de la recherche biomédicale, y compris la protéomique et la biologie structurale. Cette technologie de purification des protéines, qui repose sur la fusion de protéines recombinantes à une courte Strep-tag peptide, a évolué avec l&#39;avènement des matrices d&#39;affinité portant des Strep-Tactin, une variante génétiquement modifiée de la streptavidine avec une meilleure capacité de liaison au peptide 1, 2. Les protéines de fusion contenant deux copies de Strep-tag II, désignés double-Strep-tag ou SIII-tag, présentent une plus grande affinité pour les matrices Strep-Tactin que ceux contenant seulement un Strep-tag unique, assurant la purification plus efficace des protéines recombinantes et leurs partenaires de liaison associés. Cependant, la plus grande affinité de protéines double-Strep-marqués à Strep-Tactin a aussi son revers. Élution compétitive de ces protéines avec un excès de biotine peut être incomplète, conduisant à une diminution du rendement en protéines cible. A mefficace minerai alternative est élution avec du SDS, mais elle conduit à la contamination de l&#39;échantillon non désiré avec Strep-Tactin libéré de la résine, ce qui rend le dosage incompatible avec l&#39;analyse protéomique. Cet article présente une technique pour surmonter cette limitation en première stabiliser le tétramère de résine couplée de Strep-Tactin par réticulation chimique, puis en utilisant SDS pour éluer les protéines bi-Strep-marqués et leurs complexes associés de la résine réticulée résultant. Ainsi, le rendement en protéines suffisante peut être obtenue sans contamination de l&#39;échantillon avec Strep-Tactin, ce qui permet en outre l&#39;analyse par spectrométrie de masse. La méthode est adaptée pour la purification de toute protéine de fusion recombinante avec un SIII-tag exposée en surface 3 ou bi-Strep-tag (WSHPQFEK de séquence d&#39;acides aminés (GGGS) 3 WSHPQFEK et SAWSHPQFEK (GGGS) 2 GGSAWSHPQFEK, respectivement). La protéine peut être d&#39;origine animale, végétale ou bactérienne et peut être soit isolé à partir de cellules totaleslysat ou fraction de organite enrichi. A titre d&#39;exemple, nous décrivons ici la purification d&#39;une protéine à étiquette SIII VPg-Pro Potato Virus de A (PVA) 4 à partir de la fraction nucléaire de plants de Nicotiana benthamiana infectés par le PVA. La fraction nucléaire a été isolé comme décrit précédemment 5, avec les modifications suivantes: les cellules sont traitées avec du formaldéhyde, le butyrate de sodium a été substitué dans tous les tampons avec du fluorure de sodium 5 mM, inhibiteur complet de protease a été substitué par PMSF, Triton X-100 à une concentration dans l&#39;extraction tampon N ° 2 a été abaissée à 0,3% (v / v) et le culot nucléaire obtenu par centrifugation à travers des coussins de saccharose (tampon d&#39;extraction N ° 3) a été remis en suspension dans 1,45 ml de tampon de liaison pré-réfrigérés et mis en rotation pendant 1,5 heures à 4 ° C. L&#39;extrait nucléaire résultant contenant la protéine appât SIII marqués et complexes associés (échantillon de protéine appât) a été traitée selon le protocole décrit ci-dessous (voir la section 2).

<table border="0" cellpadding="0" cellspacing="0" width="606">
	<tbody>
		<tr>
			<td>Bis(sulfosuccinimidyl) suberate (BS3), No-Weigh format, 8 x 2mg</td>
			<td>Pierce/Thermo Scientific</td>
			<td>21585</td>
			<td><a href="http://www.fishersci.com">www.fishersci.com CAUTION: Hazardous substance. Causes serious respiratory, skin and eye irritation. Wear protective gloves and eye protection.</a></td>
		</tr>
		<tr>
			<td>Strep-Tactin MacroPrep resin (50% suspension)</td>
			<td>IBA</td>
			<td>2-1505</td>
			<td><a href="http://www.iba-lifesciences.com/">www.iba-lifesciences.com</a></td>
		</tr>
		<tr>
			<td>Spin-X centrifuge tube filter, cellulose acetate membrane, pore size 0.45 &#956;m, non-sterile</td>
			<td>Costar (Corning)</td>
			<td>8163</td>
			<td><a href="http://www.corning.com/lifesciences/">www.corning.com/lifesciences/</a></td>
		</tr>
		<tr>
			<td>Dolphin-nose tubes</td>
			<td>Costar (Corning)</td>
			<td>3213</td>
			<td><a href="http://www.corning.com/lifesciences/">www.corning.com/lifesciences/</a></td>
		</tr>
		<tr>
			<td>Avidin&#160;</td>
			<td>IBA</td>
			<td>2-0204</td>
			<td><a href="http://www.iba-lifesciences.com/">www.iba-lifesciences.com</a></td>
		</tr>
	</tbody>
</table>

one-step purification of twin-strep-tagged proteins and their complexes on strep-tactin resin cross-linked with bis(sulfosuccinimidyl) suberate (bs3)

La purification par affinité de protéines de fusion Strep-marquées sur résines portant une streptavidine ingénierie (Strep-Tactin) est devenue une méthode largement utilisée pour l&#39;isolement des complexes de protéines dans des conditions physiologiques. Les protéines de fusion contenant deux copies de Strep-tag II, désignés double-Strep-tag ou SIII-tag, ont l&#39;avantage d&#39;une plus grande affinité pour Strep-Tactin par rapport à celles ne contenant qu&#39;un Strep-tag unique, permettant ainsi la purification de protéines plus efficace. Cependant, cet avantage est compensé par le fait que l&#39;élution de protéines double Strep-marqués avec de la biotine peut être incomplète, conduisant à une faible récupération des protéines. La récupération peut être considérablement améliorée en utilisant une élution avec dénaturant le dodécylsulfate de sodium (SDS), mais ceci conduit à une contamination par des Strep-Tactin libéré de la résine de l&#39;échantillon, rendant le test incompatible avec l&#39;analyse protéomique aval. Pour surmonter cette limitation, nous avons mis au point un procédé par lequel des tétramères de résine couplée à de Strep-Tactinest tout d&#39;abord stabilisé par des liaisons covalentes de réticulation avec Bis (sulfosuccinimidyl) subérate (BS3) et de la résine réticulée résultante est ensuite utilisée pour purifier des complexes de protéines cibles en une seule étape de purification par lots. Élution efficace avec SDS assure une bonne récupération des protéines, tandis que l&#39;absence de contamination Strep-Tactin permet en aval l&#39;analyse des protéines par spectrométrie de masse. Comme une preuve de concept, que nous décrivons ici un protocole pour la purification de la protéine virale SIII étiqueté VPg-Pro à partir de noyaux de N. infecté par un virus benthamiana plantes à l&#39;aide de la résine de polyméthacrylate de Strep-Tactin réticulé avec BS3. Le même protocole peut être utilisé pour purifier n&#39;importe quelle protéine bi-Strep-marqué d&#39;intérêt et caractériser ses partenaires de liaison physiologiques.

La procédure de purification est illustrée schématiquement sur ​​la figure 1, avec une représentation des problèmes associés à d&#39;autres procédés de purification existants. <img alt="Figure 1" fo:content-width="5.5in" src="/files/ftp_upload/51536/51536fig1highres.jpg" width="650" /> Figure 1. Représentation schématique de la procédure de purification. Le flux de travail comprend la stabilisation d...

Watch this Scientific Journal Video about One-step Purification of Twin-Strep-tagged Proteins and Their Complexes on Strep-Tactin Resin Cross-linked With Bissulfosuccinimidyl Suberate BS3 at JoVE.com

One-step Purification of Twin-Strep-tagged Proteins and Their Complexes on Strep-Tactin Resin Cross-linked With Bissulfosuccinimidyl Suberate BS3

Une méthode est décrite pour la purification efficace de protéines de fusion bi-Strep-marqués et leurs complexes spécifiques sur streptavidine modifiée (Strep-Tactin) résine réticulé de manière covalente avec Bis (sulfosuccinimidyl) subérate (BS3). Le procédé présente les avantages d&#39;une vitesse rapide, une bonne récupération de la protéine cible et de haute pureté, et est compatible avec une analyse ultérieure par spectrométrie de masse.

Une étape de purification de protéines Twin-Strep-marqués et leurs complexes sur Strep-Tactin résine réticulée Avec Bis (sulfosuccinimidyl) subérate (BS3)

Affinity purification of Strep-tagged fusion proteins on resins carrying an engineered streptavidin (Strep-Tactin) has become a widely used method for ...

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Research

JoVE Journal

Biology

One-step Purification of Twin-Strep-tagged Proteins and Their Complexes on Strep-Tactin Resin Cross-linked With Bis(sulfosuccinimidyl) Suberate (BS3)

18.6K vues.  University of Helsinki.  Une méthode est décrite pour la purification efficace de protéines de fusion bi-Strep-marqués et leurs complexes spécifiques sur streptavidine modifiée (Strep-Tactin) résine réticulé de manière covalente avec Bis (sulfosuccinimidyl) subérate (BS3). Le procédé présente les avantages d'une vitesse rapide, une bonne récupération de la protéine cible et de haute pureté, et est compatible avec une analyse ultérieure par spectrométrie de masse. 

Vidéo: One-step Purification of Twin-Strep-tagged Proteins and Their Complexes on Strep-Tactin Resin Cross-linked With Bissulfosuccinimidyl Suberate BS3

Purification and Visualization of Influenza A Viral Ribonucleoprotein Complexes

The influenza A viral genome consists of eight negative-sense, single stranded RNA molecules, individually packed with multiple copies of the influenza A nucleoprotein (NP) into viral ribonulceoprotein particles (vRNPs). The influenza vRNPs are enclosed within the viral envelope. During cell entry, however, these vRNP complexes are released into the cytoplasm, where they gain access to the host nuclear transport machinery. In order to study the nuclear import of influenza vRNPs and the replication of the influenza genome, it is useful to work with isolated vRNPs so that other components of the virus do not interfere with these processes. Here, we describe a procedure to purify these vRNPs from the influenza A virus. The procedure starts with the disruption of the influenza A virion with detergents in order to release the vRNP complexes from the enveloped virion. The vRNPs are then separated from the other components of the influenza A virion on a 33-70% discontinuous glycerol gradient by velocity sedimentation. The fractions obtained from the glycerol gradient are then analyzed on via SDS-PAGE after staining with Coomassie blue. The peak fractions containing NP are then pooled together and concentrated by centrifugation. After concentration, the integrity of the vRNPs is verified by visualization of the vRNPs by transmission electron microscopy after negative staining. The glycerol gradient purification is a modification of that from Kemler et al. (1994)1, and the negative staining has been performed by Wu et al. (2007).2

The genome of the influenza A virus consists of eight separate complexes of RNA and proteins, termed viral ribonucleoprotein complexes (vRNPs). This paper describes the glycerol gradient purification and transmission electron microscopy visualization of influenza A vRNPs.

Purification et visualisation de la grippe A complexes ribonucléoprotéiques virale

The influenza A viral genome consists of eight negative-sense, single stranded RNA molecules, individually packed with multiple copies of the influenza A ...

Recherche

Biologie

Identification of protein complexes with quantitative proteomics in S. cerevisiae

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as important intracellular signalling molecules. However, unlike proteins, lipids are small hydrophobic molecules that traffic primarily by poorly described nonvesicular routes, which are hypothesized to occur at membrane contact sites (MCSs). MCSs are regions where the endoplasmic reticulum (ER) makes direct physical contact with a partnering organelle, e.g., plasma membrane (PM). The ER portion of ER-PM MCSs is enriched in lipid-synthesizing enzymes, suggesting that lipid synthesis is directed to these sites and implying that MCSs are important for lipid traffic. Yeast is an ideal model to study ER-PM MCSs because of their abundance, with over 1000 contacts per cell, and their conserved nature in all eukaryotes. Uncovering the proteins that constitute MCSs is critical to understanding how lipids traffic is accomplished in cells, and how they act as signaling molecules. We have found that an ER called Scs2p localize to ER-PM MCSs and is important for their formation. We are focused on uncovering the molecular partners of Scs2p. Identification of protein complexes traditionally relies on first resolving purified protein samples by gel electrophoresis, followed by in-gel digestion of protein bands and analysis of peptides by mass spectrometry. This often limits the study to a small subset of proteins. Also, protein complexes are exposed to denaturing or non-physiological conditions during the procedure. To circumvent these problems, we have implemented a large-scale quantitative proteomics technique to extract unbiased and quantified data. We use stable isotope labeling with amino acids in cell culture (SILAC) to incorporate staple isotope nuclei in proteins in an untagged control strain. Equal volumes of tagged culture and untagged, SILAC-labeled culture are mixed together and lysed by grinding in liquid nitrogen. We then carry out an affinity purification procedure to pull down protein complexes. Finally, we precipitate the protein sample, which is ready for analysis by high-performance liquid chromatography/ tandem mass spectrometry. Most importantly, proteins in the control strain are labeled by the heavy isotope and will produce a mass/ charge shift that can be quantified against the unlabeled proteins in the bait strain. Therefore, contaminants, or unspecific binding can be easily eliminated. By using this approach, we have identified several novel proteins that localize to ER-PM MCSs. Here we present a detailed description of our approach. 

Here we describe a new quantitative proteomics technique for identifying protein complexes in Saccharomyces cerevisiae. In this study, we have used the SILAC method together with affinity purification followed by tandem mass spectrometry to identify with high specificity the binding partners of an ER protein, Scs2p.

Identification des complexes protéiques avec la protéomique quantitative chez S. cerevisiae

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as ...

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 ...

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System 

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers. Genetic studies in mammalian systems have been limited due to the lack of readily available tools including defined mutant genetic cell lines, regulatory expression systems, and appropriate selectable markers. To circumvent these difficulties, model genetic systems in lower eukaryotes have become an attractive choice for the study of functionally conserved DNA repair proteins and pathways. We have developed a model yeast system to study the poorly defined genetic functions of the Werner syndrome helicase-nuclease (WRN) in nucleic acid metabolism. Cellular phenotypes associated with defined genetic mutant backgrounds can be investigated to clarify the cellular and molecular functions of WRN through its catalytic activities and protein interactions. The human WRN gene and associated variants, cloned into DNA plasmids for expression in yeast, can be placed under the control of a regulatory plasmid element. The expression construct can then be transformed into the appropriate yeast mutant background, and genetic function assayed by a variety of methodologies. Using this approach, we determined that WRN, like its related RecQ family members BLM and Sgs1, operates in a Top3-dependent pathway that is likely to be important for genomic stability. This is described in our recent publication [1] at <a href="http://www.impactaging.com">www.impactaging.com</a>. Detailed methods of specific assays for genetic complementation studies in yeast are provided in this paper.

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System

Genetic studies in yeast can be employed to investigate the molecular and cellular functions of human genes in cellular DNA metabolism. Methods are described for the genetic characterization of the human WRN gene product defective in the premature aging disorder Werner syndrome in functionally conserved pathways using yeast as a tractable model system.

Les études génétiques des protéines humaines réparation de l&#39;ADN utilisant de la levure comme système modèle

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers.  Genetic studies in mammalian ...

One-step Metabolomics: Carbohydrates, Organic and Amino Acids Quantified in a Single Procedure

Every infant born in the US is now screened for up to 42 rare genetic disorders called "inborn errors of metabolism". The screening method is based on tandem mass spectrometry and quantifies acylcarnitines as a screen for organic acidemias and also measures amino acids. All states also perform enzymatic testing for carbohydrate disorders such as galactosemia. Because the results can be non-specific, follow-up testing of positive results is required using a more definitive method. The present report describes the "urease" method of sample preparation for inborn error screening. Crystalline urease enzyme is used to remove urea from body fluids which permits most other water-soluble metabolites to be dehydrated and derivatized for gas chromatography in a single procedure. Dehydration by evaporation in a nitrogen stream is facilitated by adding acetonitrile and methylene chloride. Then, trimethylsilylation takes place in the presence of a unique catalyst, triethylammonium trifluoroacetate. Automated injection and chromatography is followed by macro-driven custom quantification of 192 metabolites and semi-quantification of every major component using specialized libraries of mass spectra of TMS derivatized biological compounds. The analysis may be performed on the widely-used Chemstation platform using the macros and libraries available from the author. In our laboratory, over 16,000 patient samples have been analyzed using the method with a diagnostic yield of about 17%--that is, 17% of the samples results reveal findings that should be acted upon by the ordering physician. Included in these are over 180 confirmed inborn errors, of which about 38% could not have been diagnosed using previous methods.

The urease method of sample preparation for GC/MS analysis of intermediary metabolites is presented by its inventor. The method allows one-step follow-up of newborn screening for inborn errors by tandem mass spectrometry by quantifying carbohydrates, organic and amino acids all in a single process.

Une étape métabolomique: glucides, acides organiques et aminés quantifiés dans une procédure unique

Every infant born in the US is now screened for up to 42 rare genetic disorders called "inborn errors of metabolism". The screening method is based on ...

Preparation, Purification, and Characterization of Lanthanide Complexes for Use as Contrast Agents for Magnetic Resonance Imaging

Polyaminopolycarboxylate-based ligands are commonly used to chelate lanthanide ions, and the resulting complexes are 
useful as contrast agents for magnetic resonance imaging (MRI). Many commercially available ligands are especially useful because they contain 
functional groups that allow for fast, high-purity, and high-yielding conjugation to macromolecules and biomolecules via amine-reactive 
activated esters and isothiocyanate groups or thiol-reactive maleimides. While metalation of these ligands is considered common 
knowledge in the field of bioconjugation chemistry, subtle differences in metalation procedures must be taken into account when 
selecting metal starting materials. Furthermore, multiple options for purification and characterization exist, and selection of the most 
effective procedure partially depends on the selection of starting materials. These subtle differences are often neglected in published 
protocols. Here, our goal is to demonstrate common methods for metalation, purification, and characterization of lanthanide complexes 
that can be used as contrast agents for MRI (Figure 1). We expect that this publication will enable biomedical scientists to incorporate 
lanthanide complexation reactions into their repertoire of commonly used reactions by easing the selection of starting materials and 
purification methods.

We demonstrate the metalation, purification, and characterization of lanthanide complexes. The complexes described here can be conjugated to macromolecules to enable tracking of these molecules using magnetic resonance imaging.

Préparation, la purification et la caractérisation des complexes de lanthanides pour utilisation comme agents de contraste pour imagerie par résonance magnétique

Polyaminopolycarboxylate-based ligands are commonly used to chelate lanthanide ions, and the resulting complexes are 
useful as contrast agents for ...

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems1, 2. Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria1-6. In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes1, 2, 7. Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast8, 9, and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system10. Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large-scale cultures. Tandem mass spectrometry is then used to identify the stably co-purifying proteins with high sensitivity (low nanogram detection limits).
Here, we describe detailed step-by-step procedures we commonly use for systematic protein tagging, purification and mass spectrometry-based analysis of soluble protein complexes from E. coli, which can be scaled up and potentially tailored to other bacterial species, including certain opportunistic pathogens that are amenable to recombineering. The resulting physical interactions can often reveal interesting unexpected components and connections suggesting novel mechanistic links. Integration of the PPI data with alternate molecular association data such as genetic (gene-gene) interactions and genomic-context (GC) predictions can facilitate elucidation of the global molecular organization of multi-protein complexes within biological pathways. The networks generated for E. coli can be used to gain insight into the functional architecture of orthologous gene products in other microbes for which functional annotations are currently lacking.

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.

Identification des complexes protéiques dans<em&gt; Escherichia coli</em&gt; En utilisant séquentielle Purification par affinité peptide en combinaison avec la spectrométrie de masse en tandem

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the ...

Formulations for Freeze-drying of Bacteria and Their Influence on Cell Survival

Cellular water can be removed to reversibly inactivate microorganisms to facilitate storage. One such method of removal is freeze-drying, which is considered a gentle dehydration method. To facilitate cell survival during drying, the cells are often formulated beforehand. The formulation forms a matrix that embeds the cells and protects them from various harmful stresses imposed on the cells during freezing and drying. We present here a general method to evaluate the survival rate of cells after freeze-drying and we illustrate it by comparing the results obtained with four different formulations: the disaccharide sucrose, the sucrose derived polymer Ficoll PM400, and the respective polysaccharides hydroxyethyl cellulose (HEC) and hydroxypropyl methyl cellulose (HPMC), on two strains of bacteria, P. putida KT2440 and A. chlorophenolicus A6. In this work we illustrate how to prepare formulations for freeze-drying and how to investigate the mechanisms of cell survival after rehydration by characterizing the formulation using of differential scanning calorimetry (DSC), surface tension measurements, X-ray analysis, and electron microscopy and relating those data to survival rates. The polymers were chosen to get a monomeric structure of the respective polysaccharide resembling sucrose to a varying degrees. Using this method setup we showed that polymers can support cell survival as effectively as disaccharides if certain physical properties of the formulation are controlled1.

Freeze-drying is often an easy and convenient way to obtain dry products of viable bacterial cells. An issue of the process is cell survival. We detail here a procedure to investigate how cell survival during freeze-drying is influenced by the properties of the formulation used.

Formulations pour la lyophilisation des bactéries et leur influence sur la survie des cellules

Cellular water can be removed to reversibly inactivate microorganisms to facilitate storage. One such method of removal is freeze-drying, which is ...

High-throughput Purification of Affinity-tagged Recombinant Proteins

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further biochemical analyses to obtain detailed insights into structure/function relationships. Advances in oligonucleotide- and gene synthesis technology make large-scale mutagenesis strategies increasingly feasible, including the substitution of target residues by all 19 other amino acids. Gain- or loss-of-function phenotypes then allow systematic conclusions to be drawn, such as the contribution of particular residues to catalytic activity, protein stability and/or protein-protein interaction specificity.
In order to attribute the different phenotypes to the nature of the mutation - rather than to fluctuating experimental conditions - it is vital to purify and analyse the proteins in a controlled and reproducible manner. High-throughput strategies and the automation of manual protocols on robotic liquid-handling platforms have created opportunities to perform such complex molecular biological procedures with little human intervention and minimal error rates1-5.
Here, we present a general method for the purification of His-tagged recombinant proteins in a high-throughput manner. In a recent study, we applied this method to a detailed structure-function investigation of TFIIB, a component of the basal transcription machinery. TFIIB is indispensable for promoter-directed transcription in vitro and is essential for the recruitment of RNA polymerase into a preinitiation complex6-8. TFIIB contains a flexible linker domain that penetrates the active site cleft of RNA polymerase9-11. This linker domain confers two biochemically quantifiable activities on TFIIB, namely (i) the stimulation of the catalytic activity during the 'abortive' stage of transcript initiation, and (ii) an additional contribution to the specific recruitment of RNA polymerase into the preinitiation complex4,5,12 . We exploited the high-throughput purification method to generate single, double and triple substitution and deletions mutations within the TFIIB linker and to subsequently analyse them in functional assays for their stimulation effect on the catalytic activity of RNA polymerase4. Altogether, we generated, purified and analysed 381 mutants - a task which would have been time-consuming and laborious to perform manually. We produced and assayed the proteins in multiplicates which allowed us to appreciate any experimental variations and gave us a clear idea of the reproducibility of our results.
This method serves as a generic protocol for the purification of His-tagged proteins and has been successfully used to purify other recombinant proteins. It is currently optimised for the purification of 24 proteins but can be adapted to purify up to 96 proteins.

We describe a method for the affinity-tagged purification of recombinant proteins using liquid-handling robotics. This method is generally applicable to the small-scale purification of soluble His-tagged proteins in a high-throughput format.

À haut débit Affinity Purification de protéines recombinantes-étiquette

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further ...

Purification of Transcripts and Metabolites from Drosophila Heads

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. Although fruit flies provide obvious experimental advantages, research on neurodegenerative diseases has mostly relied on traditional techniques, including genetic interaction, histology, immunofluorescence, and protein biochemistry. These techniques are effective for mechanistic, hypothesis-driven studies, which lead to a detailed understanding of the role of single genes in well-defined biological problems. However, neurodegenerative diseases are highly complex and affect multiple cellular organelles and processes over time. The advent of new technologies and the omics age provides a unique opportunity to understand the global cellular perturbations underlying complex diseases. Flexible model organisms such as Drosophila are ideal for adapting these new technologies because of their strong annotation and high tractability. One challenge with these small animals, though, is the purification of enough informational molecules (DNA, mRNA, protein, metabolites) from highly relevant tissues such as fly brains. Other challenges consist of collecting large numbers of flies for experimental replicates (critical for statistical robustness) and developing consistent procedures for the purification of high-quality biological material. Here, we describe the procedures for collecting thousands of fly heads and the extraction of transcripts and metabolites to understand how global changes in gene expression and metabolism contribute to neurodegenerative diseases. These procedures are easily scalable and can be applied to the study of proteomic and epigenomic contributions to disease.

Purification of Transcripts and Metabolites from Drosophila Heads

We describe here the procedures for the extraction and purification of mRNA and metabolites from Drosophila heads. We are applying these techniques to better understand the cellular perturbations underlying neuronal degeneration. These methodologies can be easily scaled and adapted for other "omic" projects.

Purification des relevés de notes et de métabolites<em&gt; Drosophila</em&gt; Têtes

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. ...