{"id":25,"date":"2020-05-26T22:43:00","date_gmt":"2020-05-26T22:43:00","guid":{"rendered":""},"modified":"2022-09-28T20:20:57","modified_gmt":"2022-09-28T20:20:57","slug":"methodes-electrophoretiques-principe-appareillage-et-application-en-domaine-medicale","status":"publish","type":"post","link":"https:\/\/avo6.com\/?p=25","title":{"rendered":"METHODES ELECTROPHORETIQUES : PRINCIPE, APPAREILLAGE ET APPLICATION EN DOMAINE MEDICALE"},"content":{"rendered":"
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DEFINITION<\/span><\/span><\/span><\/b><\/div>\n
Electrophor\u00e8se: terme d\u2019origine grecque signifiant transport ou anciennement cataphor\u00e8se.<\/span><\/div>\n
L\u2019\u00e9lectrophor\u00e8se est un processus de migration des mol\u00e9cules charg\u00e9es (phase solide), dans un milieu liquide tamponn\u00e9, sous l\u2019influence d\u2019un courant \u00e9lectrique continu.<\/span><\/div>\n
Elle permet de s\u00e9parer, de doser et d\u2019identifier les constituants d\u2019un m\u00e9lange en solution en se fondant sur la diff\u00e9rence de migration de ses composants, lorsqu\u2019ils sont soumis \u00e0 un champ \u00e9lectrique.<\/span><\/div>\n
Apr\u00e8s migration, les diff\u00e9rentes prot\u00e9ines se localisent au niveau d\u2019une zone donn\u00e9e du support d\u2019o\u00f9 le nom \u00e9lectrophor\u00e8se de zone.<\/span><\/div>\n
PRINCIPE<\/span><\/span><\/span><\/b><\/div>\n
L\u2019\u00e9lectrophor\u00e8se regroupe les m\u00e9thodes de s\u00e9paration reposant sur les diff\u00e9rences de vitesse de migration des prot\u00e9ines charg\u00e9es dans une solution d\u2019\u00e9lectrolytes sous l\u2019influence d\u2019un champ \u00e9lectrique sur un support donn\u00e9.<\/span><\/div>\n
Les mol\u00e9cules charg\u00e9es (-) anions: migrent vers l\u2019anode (+)<\/span><\/div>\n
Les mol\u00e9cules charg\u00e9es (+) cations: migrent vers la cathode (-)<\/span><\/div>\n
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Les prot\u00e9ines sont s\u00e9par\u00e9es en fractions de mobilit\u00e9 diff\u00e9rente,<\/span><\/div>\n
Les techniques de r\u00e9v\u00e9lation sont nombreuses pour quantifier ces prot\u00e9ines<\/span><\/div>\n
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ETUDE THEORIQUE DU DEPLACEMENT ELECTROPHORETIQUE<\/span><\/span><\/span><\/b><\/div>\n<\/div>\n
1.\u00a0\u00a0\u00a0\u00a0\u00a0<\/span><\/span><\/span><\/b><\/span>Origine des charges<\/span><\/span><\/b>
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Les substances ayant des groupes fonctionnels capables de s’ioniser en solution donnent naissance \u00e0 des particules charg\u00e9es positivement ou n\u00e9gativement.<\/span><\/div>\n
Cette ionisation d\u00e9pend non seulement de la nature m\u00eame des compos\u00e9s, mais \u00e9galement du milieu\u00a0\u00a0<\/span>environnant qui intervient par sa composition, son pH et sa force ionique.\u00a0<\/span><\/span><\/div>\n
On d\u00e9finit le point iso\u00e9lectrique (pHi) comme la valeur du pH pour lequel il n’y a aucune migration \u00e9lectrophor\u00e9tique (l\u2019ensemble des charges est nul).\u00a0<\/span><\/span><\/div>\n
\u00ae<\/span>Seule la charge nette<\/span>\u00a0\u00a0<\/span><\/span>Q<\/span><\/b>\u00a0d’une particule intervient dans son d\u00e9placement (mobilit\u00e9 \u00e9lectrophor\u00e9tique).<\/span><\/div>\n
La charge nette Q est variable selon le pH de la solution dans laquelle elle est dilu\u00e9e,<\/span><\/div>\n
\u00fc\u00a0\u00a0<\/span><\/span><\/span>Si pH &gt; pHi, la prot\u00e9ine a une charge globale n\u00e9gative; dans un champ \u00e9lectrique, elle se d\u00e9place vers l\u2019\u00e9lectrode + (anode)<\/span><\/div>\n
\u00fc\u00a0\u00a0<\/span><\/span><\/span>Si pH &lt; pHi, la prot\u00e9ine a une charge globale positive; dans un champ \u00e9lectrique, elle se d\u00e9place vers l\u2019\u00e9lectrode – (cathode)<\/span><\/div>\n
\u00fc\u00a0\u00a0<\/span><\/span><\/span>Si pH = pHi, la prot\u00e9ine est sous forme d\u2019un zwitterion (neutre); elle ne se d\u00e9place pas sous l\u2019influence d\u2019un champ \u00e9lectrique<\/span>
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2.\u00a0\u00a0\u00a0\u00a0\u00a0<\/span><\/span><\/span><\/b><\/span>Mobilit\u00e9 \u00e9lectrophor\u00e9tique\u00a0: \u00b5<\/span><\/span><\/b>
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Chaque particule a une\u00a0<\/span>\u00b5<\/span><\/b>\u00a0sp\u00e9cifique.<\/span><\/div>\n
Si 2 \u00e9lectrodes sont reli\u00e9es \u00e0 un g\u00e9n\u00e9rateur de courant continu et qu’une certaine tension est impos\u00e9e, il se cr\u00e9e entre ces 2 \u00e9lectrodes un champ \u00e9lectrique E capable d’exercer une certaine force\u00a0\u00a0<\/span>F sur un granule que l’on suppose sph\u00e9rique de rayon r (ionis\u00e9e \u00e0 charge nette\u00a0<\/span>Q<\/span><\/b>).<\/span><\/div>\n
Cette force provoque la migration de la particule ionis\u00e9e vers l’\u00e9lectrode de charge oppos\u00e9e avec une certaine vitesse\u00a0V<\/b>.<\/span><\/div>\n
F=E x Q<\/span><\/div>\n
Par opposition \u00e0 cette force F, il y a F\u2019\u00a0: force de frottement\u00a0\u00a0<\/span>due \u00e0 la viscosit\u00e9 du milieu (\u00e9ta) \u03b7 et elle va s’opposer \u00e0 la migration de la particule et la freiner.<\/span><\/div>\n
F’=6 \u03b7 r\u00a0<\/span>\u03c0<\/span>\u00a0v<\/span><\/div>\n
V : vitesse de la particule exprime en cm\/seconde<\/span><\/div>\n
Mobilit\u00e9 \u00e9lectrophor\u00e9tique\u00a0<\/span>\u00b5<\/span><\/b>\u00a0<\/span><\/span><\/div>\n
Formule: u = v \/ E= Q\/6 \u03b7 r\u00a0<\/span>\u03c0<\/span><\/div>\n
Chaque esp\u00e8ce de mol\u00e9cules ayant des mobilit\u00e9s diff\u00e9rentes, il est possible en se basant sur cette propri\u00e9t\u00e9 d’effectuer leur s\u00e9paration.<\/span><\/div>\n
3.\u00a0\u00a0\u00a0\u00a0\u00a0<\/span><\/span><\/span><\/b><\/span>Le support<\/span><\/span><\/b>
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Quelle que soit sa nature, le support poss\u00e8de un pouvoir adsorbant capable de ralentir les migrations.\u00a0<\/span><\/span><\/div>\n
\u00a0<\/span>Il faut noter que les sinuosit\u00e9s\u00a0\u00a0<\/span>dans les\u00a0\u00a0<\/span>canalicules du support font\u00a0\u00a0<\/span>appara\u00eetre une distance parcourue plus petite que la distance r\u00e9ellement parcourue \u2192 \u00e9talement des bande de s\u00e9paration.<\/span><\/div>\n
4.\u00a0\u00a0\u00a0\u00a0\u00a0<\/span><\/span><\/span><\/b><\/span>Le champ \u00e9lectrique<\/span><\/span><\/b>
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Le d\u00e9placement \u00e9lectrophor\u00e9tique est proportionnel\u00a0:<\/span><\/div>\n
\u00fc\u00a0\u00a0<\/span><\/span><\/span>\u00e0\u00a0\u00a0<\/span>la mobilit\u00e9 de la particule consid\u00e9r\u00e9e,<\/span><\/div>\n
\u00fc\u00a0\u00a0<\/span><\/span><\/span>au champ \u00e9lectrique E<\/span><\/div>\n
\u00fc\u00a0\u00a0<\/span><\/span><\/span>\u00a0<\/span>au temps pendant lequel ce champ est impos\u00e9.\u00a0<\/span><\/span><\/div>\n
La mobilit\u00e9 \u00e9tant une caract\u00e9ristique de la particule consid\u00e9r\u00e9e, donc pour obtenir une meilleure s\u00e9paration, il faudra agir sur le champ \u00e9lectrique et le temps.<\/span><\/div>\n
5.\u00a0\u00a0\u00a0\u00a0\u00a0<\/span><\/span><\/span><\/b><\/span>La force ionique\u00a0: Fi<\/span><\/span><\/b><\/div>\n
Elle intervient sur la valeur du champ \u00e9lectrique et sur la mobilit\u00e9 des compos\u00e9s.\u00a0<\/span><\/span><\/div>\n
6.\u00a0\u00a0\u00a0\u00a0\u00a0<\/span><\/span><\/span><\/b><\/span>Le pH<\/span><\/span><\/b>
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L’action du pH est pr\u00e9pond\u00e9rante pour les compos\u00e9s organiques ayant des groupements acides faibles ou bases faibles,\u00a0<\/span><\/span><\/div>\n
Ainsi que pour les compos\u00e9s amphot\u00e8res, il est stabilis\u00e9 en utilisant des solutions tampons.<\/span><\/div>\n
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\u00a0<\/span><\/b>D\u00c9PLACEMENTS<\/b><\/span>\u00a0DUS A DES <\/b>PH\u00c9NOM\u00c8NES<\/b><\/span>\u00a0SECONDAIRES<\/b><\/span><\/span><\/span><\/div>\n
1.\u00a0\u00a0\u00a0\u00a0\u00a0<\/span><\/span><\/span><\/b><\/span>Le mouvement d’\u00e9lectroendosmose<\/span><\/span><\/b>
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Le liquide contigu aux mol\u00e9cules en d\u00e9placement est charg\u00e9 en sens contraire.\u00a0<\/span><\/span><\/div>\n
Il se d\u00e9place donc dans le sens oppos\u00e9 au courant \u00e9lectrique.\u00a0<\/span><\/span><\/div>\n
Le mouvement d’\u00e9lectroendosmose<\/span><\/b>\u00a0int\u00e9resse l’ensemble de la solution d’\u00e9lectrolytes, impr\u00e9gnant le support, qui se d\u00e9place toute enti\u00e8re vers le p\u00f4le n\u00e9gatif.\u00a0<\/span><\/span><\/div>\n
L’amplitude du d\u00e9placement d\u00fb \u00e0 l’\u00e9lectroendosmose d\u00e9pend de\u00a0:<\/span><\/div>\n
\u00fc\u00a0\u00a0<\/span><\/span><\/span>\u00a0<\/span>la nature du support<\/span><\/div>\n
\u00fc\u00a0\u00a0<\/span><\/span><\/span>la force ionique<\/span><\/div>\n
\u00fc\u00a0\u00a0<\/span><\/span><\/span>\u00a0<\/span>pH du tampon.<\/span>
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2.\u00a0\u00a0\u00a0\u00a0\u00a0<\/span><\/span><\/span><\/b><\/span>\u00a0<\/span>Ph\u00e9nom\u00e8nes d’\u00e9vaporation<\/span><\/span><\/b>
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Le passage du courant \u00e9lectrique entra\u00eene par effet Joule, un d\u00e9gagement de chaleur qui provoque l\u2019’\u00e9vaporation de la solution d’\u00e9lectrolytes\u00a0<\/span>\u00e0<\/span>on a ainsi une modification du la migration \u00e9lectrophor\u00e9tique.<\/span><\/div>\n
3.\u00a0\u00a0\u00a0\u00a0\u00a0<\/span><\/span><\/span><\/b><\/span>Ph\u00e9nom\u00e8nes de diffusion<\/span><\/span><\/b>
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Conform\u00e9ment \u00e0 la loi de Fick, les particules ont tendance \u00e0 diffuser des r\u00e9gions o\u00f9 elles sont \u00e0 la plus forte concentration vers les concentrations plus faibles.\u00a0<\/span><\/span><\/div>\n
4.\u00a0\u00a0\u00a0\u00a0\u00a0<\/span><\/span><\/span><\/b><\/span>\u00c9lectrolyse<\/span><\/span><\/b>
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Ce courant s’\u00e9tablit lorsqu’il y a modification de la composition du tampon dans les compartiments des \u00e9lectrodes \u00e0 la suite de la d\u00e9charge des ions sur les \u00e9lectrodes.<\/span><\/div>\n
APPAREILLAGE<\/span><\/span><\/span><\/b><\/div>\n
Compte tenu de la vari\u00e9t\u00e9 des appareils d’\u00e9lectrophor\u00e8se, il est impossible de tous les d\u00e9crire. On retrouve cependant, toujours 3 \u00e9l\u00e9ments principaux<\/span><\/div>\n
\u00fc\u00a0\u00a0<\/span><\/span><\/span>le g\u00e9n\u00e9rateur,\u00a0<\/span><\/span><\/div>\n
\u00fc\u00a0\u00a0<\/span><\/span><\/span>la cuve,\u00a0<\/span><\/span><\/div>\n
\u00fc\u00a0\u00a0<\/span><\/span><\/span>les \u00e9lectrodes.<\/span><\/div>\n
\u00fc\u00a0\u00a0<\/span><\/span><\/span>Support<\/span><\/div>\n
En plus\u00a0: Support, solution tampon.<\/span>
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1.\u00a0\u00a0\u00a0\u00a0\u00a0<\/span><\/span><\/span><\/b><\/span>Le g\u00e9n\u00e9rateur<\/span><\/span><\/b><\/span>
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Il\u00a0\u00a0<\/span>est reli\u00e9 aux 2 \u00e9lectrodes de la cuve \u00e9lectrophor\u00e9tique. On utilise un courant\u00a0:<\/span><\/div>\n
\u00fc\u00a0\u00a0<\/span><\/span><\/span>Soit en tension stabilis\u00e9e\u00a00 &lt; V &lt; 500V<\/span><\/span><\/div>\n
\u00fc\u00a0\u00a0<\/span><\/span><\/span>Soit \u00e0 intensit\u00e9 stabilis\u00e9e\u00a0U&lt; 1 &lt; 200 Ma<\/span><\/span><\/div>\n
\u00fc\u00a0\u00a0<\/span><\/span><\/span>Soit \u00e0 puissance constante\u00a0\u00a0<\/span>0 &lt; P &lt; 400 W<\/span><\/span><\/div>\n
Le choix se fera en fonction de :<\/span><\/div>\n
\u00fc\u00a0\u00a0<\/span><\/span><\/span>La technique \u00e9lectrophor\u00e9tique<\/span><\/div>\n
\u00fc\u00a0\u00a0<\/span><\/span><\/span>\u00a0<\/span>la nature du support (\u00e9vaporation ou non)<\/span>
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2.\u00a0\u00a0\u00a0\u00a0\u00a0<\/span><\/span><\/span><\/b><\/span>La cuve<\/span><\/span><\/b>
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Les cuves doivent \u00eatre ferm\u00e9es et satur\u00e9es en vapeur de solvant, de fa\u00e7on \u00e0 minimiser les ph\u00e9nom\u00e8nes\u00a0\u00a0<\/span>d’\u00e9vaporation par effet Joule.\u00a0<\/span><\/span><\/div>\n
Les \u00e9lectrophor\u00e8ses sur papier ou ac\u00e9tate de cellulose se pratiquent \u00e0 l’aide de cuves dans lesquelles les supports sont maintenus \u00e0 l’horizontale.\u00a0<\/span><\/span><\/div>\n
Les \u00e9lectrophor\u00e8ses en gel de polyacrylamide et\u00a0\u00a0<\/span>les focalisations iso\u00e9lectriques peuvent se pratiquer soit\u00a0:<\/span><\/div>\n
\u00fc\u00a0\u00a0<\/span><\/span><\/span>\u00a0<\/span>en plaques horizontales<\/span><\/div>\n
\u00fc\u00a0\u00a0<\/span><\/span><\/span>\u00a0<\/span>en plaques verticales.<\/span><\/div>\n
\u00fc\u00a0\u00a0<\/span><\/span><\/span>Ou encore \u00e0 l\u2019int\u00e9rieur d\u2019un\u00a0\u00a0<\/span>tube en\u00a0\u00a0<\/span>verre.<\/span>
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3.\u00a0\u00a0\u00a0\u00a0\u00a0<\/span><\/span><\/span><\/b><\/span>Les \u00e9lectrodes<\/span><\/span><\/b>
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Elles sont en\u00a0:<\/span><\/div>\n
\u00fc\u00a0\u00a0<\/span><\/span><\/span>Graphite<\/span><\/div>\n
\u00fc\u00a0\u00a0<\/span><\/span><\/span>\u00a0<\/span>ou en platine.<\/span><\/div>\n
Afin d’\u00e9tablir un circuit \u00e9lectrique ferm\u00e9, on relie les \u00e9lectrodes entre elles par une solution conductrice d’ions (tampon).<\/span><\/div>\n
4.\u00a0\u00a0\u00a0\u00a0\u00a0<\/span><\/span><\/span><\/b><\/span>Support\u00a0:<\/span><\/span><\/b>
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Ac\u00e9tate de cellulose\u00a0:<\/span><\/b>\u00a0s\u00e9paration grossi\u00e8re (prot\u00e9ines)<\/span><\/div>\n
Papier (amidon)\u00a0:<\/span><\/b>\u00a0abandonn\u00e9 car d\u00e9p\u00f4ts+++ (aa et petites mol\u00e9cules)<\/span><\/div>\n
PAA\u00a0:<\/span><\/b>\u00a0s\u00e9pare les prot\u00e9ines et acides nucl\u00e9iques (support \u00e9lectriquement inerte)<\/span><\/div>\n
Agarose\u00a0:<\/span><\/b>\u00a0se g\u00e9lifie \u00e0 bord\u00a0: grande pr\u00e9cision (prot\u00e9ines, ac. nucl\u00e9ique, mol\u00e9cules de grande taille)<\/span>
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TECHNIQUES ELECTROPHORETIQUES\u00a0<\/span><\/span><\/span><\/span><\/b><\/div>\n
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\u00a7\u00a0\u00a0<\/span><\/span><\/span>\u00c9lectrophor\u00e8se en veine liquide\u00a0:<\/span><\/b>\u00a0Ou \u00e9lectrophor\u00e8se de fronti\u00e8re (A titre historique)<\/span><\/div>\n
\u00a7\u00a0\u00a0<\/span><\/span><\/span>\u00c9lectrophor\u00e8se en milieu h\u00e9t\u00e9rog\u00e8ne\u00a0:<\/span><\/b>
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1.\u00a0\u00a0\u00a0\u00a0\u00a0<\/span><\/span><\/span><\/b><\/span>Electrophor\u00e8se de zone ou sur support) +++<\/span><\/span><\/b>
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L’\u00e9lectrophor\u00e8se de zone ou sur support est d’un usage quotidien en biologie clinique pour l’\u00e9tude des fractions prot\u00e9iques du s\u00e9rum.<\/span><\/div>\n
Le milieu liquide dans lequel s’effectue la migration est stabilis\u00e9 par un support de nature vari\u00e9 sur\u00a0\u00a0<\/span>lequel il est ensuite plus ais\u00e9 de localiser les diff\u00e9rentes zones de migration.\u00a0<\/span><\/span><\/div>\n
Ce proc\u00e9d\u00e9 permet d’obtenir d’excellentes s\u00e9parations : ne n\u00e9cessite\u00a0\u00a0<\/span>que de tr\u00e8s faibles quantit\u00e9s d’\u00e9chantillons.<\/span><\/div>\n
Nature du support<\/span><\/div>\n
\u00fc\u00a0\u00a0<\/span><\/span><\/span>ac\u00e9tate de cellulose<\/span><\/div>\n
\u00fc\u00a0\u00a0<\/span><\/span><\/span>Agarose<\/span><\/div>\n
\u00fc\u00a0\u00a0<\/span><\/span><\/span>gels de polyacrylamide<\/span>
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2.\u00a0\u00a0\u00a0\u00a0\u00a0<\/span><\/span><\/span><\/b><\/span>\u00c9lectrophor\u00e8se en gradient de densit\u00e9<\/span><\/span><\/b>
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A l’aide d’un cr\u00e9ateur de gradient, on peut r\u00e9aliser un gel de porosit\u00e9 lin\u00e9airement croissante.\u00a0<\/span><\/span><\/div>\n
En faisant migrer un m\u00e9lange prot\u00e9ique dans un\u00a0\u00a0<\/span>tel gel, dans le sens des porosit\u00e9s d\u00e9croissantes, chaque sous unit\u00e9 prot\u00e9ique se trouvera arr\u00eat\u00e9e \u00e0 l’endroit o\u00f9 les pores du gel seront devenus trop petits pour que la prot\u00e9ine continue \u00e0 migrer.\u00a0<\/span><\/span><\/div>\n
On peut ainsi s\u00e9parer les entit\u00e9s prot\u00e9iques selon leur masse mol\u00e9culaire et augmenter la r\u00e9solution par rapport \u00e0 l’\u00e9lectrophor\u00e8se conventionnelle.<\/span><\/div>\n
3.\u00a0\u00a0\u00a0\u00a0\u00a0<\/span><\/span><\/span><\/b><\/span>Electrofocalisation ou focalisation iso\u00e9lectrique<\/span><\/span><\/b>
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Dans ce cas le PH n\u2019est pas maintenu constant dans l\u2019ensemble du syst\u00e8me, mais les <\/ins>composants de l\u2019\u00e9chantillon migrent par \u00e9lectrophor\u00e8se dans un gradient stationnaire de PH. Ainsi ils sont concentr\u00e9s \u2018focalis\u00e9s) en bandes \u00e9troites \u00e0 leur points iso\u00e9lectriques respectifs<\/span><\/div>\n
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4.\u00a0\u00a0<\/span><\/span><\/span><\/b><\/span>\u00c9lectrophor\u00e8se en gel de polyacrylamide en pr\u00e9sence de dod\u00e9cylsulfate de sodium (SDS-PAGE)<\/span><\/span><\/b>
\n
\n<\/span><\/b><\/div>\n
Le SDS combin\u00e9 \u00e0 celle d’un r\u00e9ducteur de liaisons sulfure \u00e0 temp\u00e9rature \u00e9lev\u00e9e (100\u00b0C – 1 \u00e0 2 min) dissocie (d\u00e9nature) les agr\u00e9gats prot\u00e9iques.<\/span><\/div>\n
Le SDS se fixe sur les polypeptides selon un rapport constant et les transforment en anions.\u00a0<\/span><\/span><\/div>\n
Les complexes SDS-polypeptides ont donc des charges identiques (quel que soit le polypeptide) et migrent donc exclusivement en fonction de leur taille mol\u00e9culaire.<\/span><\/div>\n
Cette technique permet donc une estimation des masses mol\u00e9culaires des peptides.<\/span><\/div>\n
5.\u00a0\u00a0\u00a0\u00a0\u00a0<\/span><\/span><\/span><\/b><\/span>\u00c9lectrophor\u00e8se bidimensionnelle<\/span><\/span><\/b>
\n
\n<\/span><\/b><\/div>\n
Divers systemes bidimensionnels ont \u00e9t\u00e9 utilis\u00e9s, le + frequent est de realiser\u00a0\u00a0<\/span>2 migrations cons\u00e9cutives dans deus directions perpendiculaires avec 2 tampons differents(par ex electrofocaalisation et electrophorese sur gel PAA-SDS)<\/span>
\n
\n<\/span><\/div>\n
6.\u00a0\u00a0\u00a0\u00a0\u00a0<\/span><\/span><\/span><\/b><\/span>\u00c9lectrophor\u00e8se capillaire<\/span><\/span><\/b><\/div>\n
Technique de s\u00e9paration \u00e9lectrocin\u00e9tique effectu\u00e9e dans un tube de diam\u00e8tre inf\u00e9rieur \u00e0 100 \u00b5m rempli d’un milieu \u00e9lectrolyte.\u00a0<\/span><\/span><\/div>\n
Les modes de d\u00e9placement des solut\u00e9s sont l’\u00e9lectromigration et l’\u00e9lectro-osmose.\u00a0<\/span><\/span><\/div>\n
Il existe 3 modes de s\u00e9paration :\u00a0<\/span><\/span><\/div>\n
\u00fc\u00a0\u00a0<\/span><\/span><\/span>Electrophor\u00e8se capillaire en solution libre o\u00f9 la s\u00e9paration se fait en fonction de leur charge au sein d’un \u00e9lectrolyte aqueux;\u00a0<\/span><\/span><\/div>\n
\u00fc\u00a0\u00a0<\/span><\/span><\/span>Chromatographie capillaire \u00e9lectrocin\u00e9tique en pr\u00e9sence de micelles o\u00f9 la s\u00e9paration se fait en fonction de leur charge et de leur hydrophobicit\u00e9 au sein d’un \u00e9lectrolyte contenant des micelles d’un surfactant;\u00a0<\/span><\/span><\/div>\n
\u00fc\u00a0\u00a0<\/span><\/span><\/span>Electrophor\u00e8se capillaire sur gel o\u00f9 les compos\u00e9s sont s\u00e9par\u00e9s au sein d’un gel (polyacrylamide par exemple).<\/span><\/div>\n
Cette technique pr\u00e9sente de nombreux avantages* :<\/span><\/div>\n
\u00fc\u00a0\u00a0<\/span><\/span><\/span>Tr\u00e8s grand pouvoir s\u00e9parateur<\/span><\/div>\n
\u00fc\u00a0\u00a0<\/span><\/span><\/span>\u00a0<\/span>Tr\u00e8s haute sensibilit\u00e9<\/span><\/div>\n
\u00fc\u00a0\u00a0<\/span><\/span><\/span>Rapidit\u00e9 d’analyse<\/span><\/div>\n
\u00fc\u00a0\u00a0<\/span><\/span><\/span>Bon aspect quantitatif des analyses<\/span>
\n
\n<\/span><\/div>\n
7.\u00a0\u00a0\u00a0\u00a0\u00a0<\/span><\/span><\/span><\/b><\/span><\/span>Electrophor\u00e8se en champs puls\u00e9\u00a0:<\/span><\/span><\/span><\/b>
\n
\n<\/span><\/b><\/div>\n
Pour r\u00e9parer les mol\u00e9cules de haut poids mol\u00e9culaire, exemple\u00a0: ADN\u00a0: changement p\u00e9riodique du sens du champs \u00e9lectrique.<\/span><\/div>\n
8.\u00a0\u00a0\u00a0\u00a0\u00a0<\/span><\/span><\/span><\/b><\/span>Techniques immuno\u00e9lectrophor\u00e9tiques<\/span><\/span><\/b>
\n
\n<\/span><\/b><\/div>\n
Elles couplent une migration \u00e9lectrophor\u00e9tique et une m\u00e9thode de pr\u00e9cipitation antig\u00e8ne- anticorps en milieu g\u00e9los\u00e9<\/span><\/div>\n
9.\u00a0\u00a0\u00a0\u00a0\u00a0<\/span><\/span><\/span><\/b><\/span>Immunofixation<\/span><\/span><\/b>
\n
\n<\/span><\/b><\/div>\n
M\u00e9thode de d\u00e9tection immunologique qui permet par pr\u00e9cipitation de mise en \u00e9vidence et de typer une Immunoglobines monoclonale dans le s\u00e9rum ou les urines d’un patient.<\/span><\/div>\n
Tend actuellement \u00e0 supplanter l’immuno\u00e9lectrophor\u00e8se\u00a0.<\/span><\/div>\n
Permet d’identifier des anticorps (des immunoglobulines monoclonales) dans un m\u00e9lange, en fonction de leur mobilit\u00e9 \u00e9lectrophor\u00e9tique. L\u2019immunofixation s\u2019effectue \u00e0 l\u2019aide d\u2019un antis\u00e9rum pentavalent.<\/span>
\n
\n<\/span><\/div>\n
TECHNIQUES DE REVELATION<\/span><\/span><\/span><\/b>
\n
\n<\/span><\/span><\/span><\/b><\/div>\n
Selon la nature des fractions \u00e0 mettre en \u00e9vidence et le support employ\u00e9, de nombreux\u00a0\u00a0<\/span>proc\u00e9d\u00e9s peuvent \u00eatre utilis\u00e9s.\u00a0<\/span><\/span><\/div>\n
La r\u00e9v\u00e9lation peut se faire par des colorants, par une m\u00e9thode physique ou apr\u00e8s transfert et empreinte.<\/span><\/div>\n
1.\u00a0\u00a0\u00a0\u00a0\u00a0<\/span><\/span><\/span><\/b><\/span>\u00a0<\/span>Impr\u00e9gnation de r\u00e9actifs colorants<\/span><\/span><\/b>
\n
\n<\/span><\/b><\/div>\n
Noir amide, rouge Ponceau, amido-schwartz …\u00a0: colorent d’une mani\u00e8re \u00e9gale les diff\u00e9rentes fractions de prot\u00e9ines.<\/span><\/div>\n
L’intensit\u00e9 des t\u00e2ches ainsi obtenues est \u00e9valu\u00e9e par d\u00e9coupage, \u00e9lution et lecture photom\u00e9trique ou par densitom\u00e9trie.<\/span><\/div>\n
2.\u00a0\u00a0\u00a0\u00a0\u00a0<\/span><\/span><\/span><\/b>Observation sous lumi\u00e8re UV\u00a0<\/span><\/span><\/b><\/span><\/div>\n
3.\u00a0\u00a0\u00a0\u00a0\u00a0<\/span><\/span><\/span><\/b>Autoradiographie directe<\/span><\/b><\/span><\/div>\n
4.\u00a0\u00a0\u00a0\u00a0\u00a0<\/span><\/span><\/span><\/b><\/span>Techniques de transfert et d’empreinte (techniques de blotting)<\/span><\/span><\/b>
\n
\n<\/span><\/span><\/b><\/div>\n
Pour le transfert, on utilise les de nylon ou accessoirement de\u00a0\u00a0<\/span>nitrocellulose qui retiennent les prot\u00e9ines ou les acide nucleiques par liaison \u00e9lectrostatique ou covalente.<\/span><\/div>\n
La r\u00e9v\u00e9lation se fait immunod\u00e9tection ou par hybridation de sonde analogue\u00a0.<\/span><\/div>\n
Cette technique s’applique aux enzymes, hormones, ADN, ARN, oncog\u00e8nes, allerg\u00e8nes<\/span>
\n
\n<\/span><\/div>\n
APPLICATIONS<\/span><\/span><\/span><\/b>
\n
\n<\/span><\/span><\/span><\/b><\/div>\n
Les applications de l’\u00e9lectrophor\u00e8se sont multiples en biologie.\u00a0<\/span><\/span><\/div>\n
Elles sont surtout qualitatives mais elles peuvent \u00eatre quantitatives\u00a0.<\/span><\/div>\n
Exemple : Si la s\u00e9paration aboutit \u00e0 des zones bien distinctes, elles peuvent \u00eatre isol\u00e9es par d\u00e9coupage\u00a0\u00a0<\/span>du support permettant le dosage (exemple : par lecture d’absorbance apr\u00e8s \u00e9lution).\u00a0<\/span><\/span><\/div>\n
1.\u00a0\u00a0\u00a0\u00a0\u00a0<\/span><\/span><\/span><\/b><\/span>\u00a0<\/span>\u00c9lectrophor\u00e8se sur ac\u00e9tate de cellulose<\/span><\/span><\/b>
\n
\n<\/span><\/span><\/b><\/div>\n
\u00fc\u00a0\u00a0<\/span><\/span><\/span>Prot\u00e9ines s\u00e9riques;\u00a0<\/span><\/span><\/div>\n
\u00fc\u00a0\u00a0<\/span><\/span><\/span>Lipoprot\u00e9ines s\u00e9riques ;\u00a0<\/span><\/span><\/div>\n
\u00fc\u00a0\u00a0<\/span><\/span><\/span>Prot\u00e9ines urinaires (apr\u00e8s concentrations) ;\u00a0<\/span><\/span><\/div>\n
\u00fc\u00a0\u00a0<\/span><\/span><\/span>Prot\u00e9ines du LCR (apr\u00e8s concentrations) ;\u00a0<\/span><\/span><\/div>\n
\u00fc\u00a0\u00a0<\/span><\/span><\/span>H\u00e9moglobines sanguines.<\/span>
\n
\n<\/span><\/div>\n
2.\u00a0\u00a0\u00a0\u00a0\u00a0<\/span><\/span><\/span><\/b><\/span>\u00c9lectrophor\u00e8se sur agarose<\/span><\/span><\/b>
\n
\n<\/span><\/b><\/div>\n
\u00fc\u00a0\u00a0<\/span><\/span><\/span>Exploration des dysprot\u00e9in\u00e9mies,\u00a0<\/span><\/span><\/div>\n
\u00fc\u00a0\u00a0<\/span><\/span><\/span>recherche des prot\u00e9ines de Bence-Jones\u00a0<\/span><\/span><\/div>\n
3.\u00a0\u00a0\u00a0\u00a0\u00a0<\/span><\/span><\/span><\/b><\/span>\u00c9lectrophor\u00e8se sur gel de polyacrylamide<\/span><\/span><\/b>
\n
\n<\/span><\/b><\/div>\n
\u00fc\u00a0\u00a0<\/span><\/span><\/span>Lipoprot\u00e9ines ;\u00a0<\/span><\/span><\/div>\n
\u00fc\u00a0\u00a0<\/span><\/span><\/span>H\u00e9moglobines sanguines;\u00a0<\/span><\/span><\/div>\n
\u00fc\u00a0\u00a0<\/span><\/span><\/span>Prot\u00e9ines du LCR ;<\/span><\/div>\n
\u00fc\u00a0\u00a0<\/span><\/span><\/span>ADN et ARN<\/span>
\n
\n<\/span><\/div>\n
4.\u00a0\u00a0\u00a0\u00a0\u00a0<\/span><\/span><\/span><\/b><\/span>Focalisation iso\u00e9lectrique<\/span><\/span><\/b>
\n
\n<\/span><\/b><\/div>\n
\u00fc\u00a0\u00a0<\/span><\/span><\/span>Haptoglobines ;\u00a0<\/span><\/span><\/div>\n
\u00fc\u00a0\u00a0<\/span><\/span><\/span>Apolipoprot\u00e9ines E ;<\/span><\/div>\n
<\/div>\n
CONCLUSION<\/span><\/span><\/span><\/b>
\n<\/span><\/span><\/span><\/b><\/div>\n
Ce sont des techniques tr\u00e8s utilis\u00e9es en routine et dans la recherche.<\/span><\/div>\n
Pr\u00e9sentent un int\u00e9r\u00eat majeur dans le diagnostic des gammapathies monoclonales, h\u00e9moglobinopathies.<\/span><\/div>\n<\/div>\n","protected":false},"excerpt":{"rendered":"

DEFINITION Electrophor\u00e8se: terme d\u2019origine grecque signifiant transport ou anciennement cataphor\u00e8se. L\u2019\u00e9lectrophor\u00e8se est un processus de migration des mol\u00e9cules charg\u00e9es (phase…<\/p>\n","protected":false},"author":2,"featured_media":143,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"om_disable_all_campaigns":false,"_monsterinsights_skip_tracking":false,"_monsterinsights_sitenote_active":false,"_monsterinsights_sitenote_note":"","_monsterinsights_sitenote_category":0},"categories":[5],"tags":[],"aioseo_notices":[],"_links":{"self":[{"href":"https:\/\/avo6.com\/index.php?rest_route=\/wp\/v2\/posts\/25"}],"collection":[{"href":"https:\/\/avo6.com\/index.php?rest_route=\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/avo6.com\/index.php?rest_route=\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/avo6.com\/index.php?rest_route=\/wp\/v2\/users\/2"}],"replies":[{"embeddable":true,"href":"https:\/\/avo6.com\/index.php?rest_route=%2Fwp%2Fv2%2Fcomments&post=25"}],"version-history":[{"count":2,"href":"https:\/\/avo6.com\/index.php?rest_route=\/wp\/v2\/posts\/25\/revisions"}],"predecessor-version":[{"id":370,"href":"https:\/\/avo6.com\/index.php?rest_route=\/wp\/v2\/posts\/25\/revisions\/370"}],"wp:featuredmedia":[{"embeddable":true,"href":"https:\/\/avo6.com\/index.php?rest_route=\/wp\/v2\/media\/143"}],"wp:attachment":[{"href":"https:\/\/avo6.com\/index.php?rest_route=%2Fwp%2Fv2%2Fmedia&parent=25"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/avo6.com\/index.php?rest_route=%2Fwp%2Fv2%2Fcategories&post=25"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/avo6.com\/index.php?rest_route=%2Fwp%2Fv2%2Ftags&post=25"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}