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en:aree:epr:altre_risorse:biacore:metodica [2022/07/27 19:31]
127.0.0.1 external edit 		en:aree:epr:altre_risorse:biacore:metodica [2024/04/19 19:52]
Gianluca Frustagli [The Sensogram] Fixed link.
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 ~~NOTOC~~ ~~NOTOC~~
  
-**Proteomics ​Area**+**EPR Area**
  
 ====== Surface Plasmon Resonance (SPR - Biacore) ====== ====== Surface Plasmon Resonance (SPR - Biacore) ======
  
 <WRAP tabs> <WRAP tabs>
-  * [[en:​aree:​proteomica:​biacore:​richiesta|Service request]] +  * [[richiesta|Service request]] 
-  * [[en:​aree:​proteomica:​biacore:​start|Abstract]] +  * [[start|Abstract]] 
-  * [[en:​aree:​proteomica:​biacore:​metodica|Method]] +  * [[metodica|Method]] 
-  * [[en:​aree:​proteomica:​biacore:​applicazioni|Applications]]+  * [[applicazioni|Applications]]
 </​WRAP>​ </​WRAP>​
  
 ==== Methodology ==== ==== Methodology ====
  
-{{ :aree:proteomica:​biacore:​metodica.png?​600 |}}+{{ :aree:epr:​altre_risorse:​biacore:​metodica.png?​600 |}}
  
 The SPR is substantially a very precise molecular scale, able to report the binding of a single amino acid (100 Da), with a sensibility of few pg/mm² in a pM range of the bulk. The SPR is substantially a very precise molecular scale, able to report the binding of a single amino acid (100 Da), with a sensibility of few pg/mm² in a pM range of the bulk.
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 ===== The SPR - BIACORE System ===== ===== The SPR - BIACORE System =====
  
-{{ :aree:proteomica:​biacore:​il_sistema_biacore.png?​600 |}}+{{ :aree:epr:​altre_risorse:​biacore:​il_sistema_biacore.png?​600 |}}
  
 **Biacore X-100**, from Cytiva, take into account this principle. **Biacore X-100**, from Cytiva, take into account this principle.
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 The experimental protocol is obtained thanks to a well suited **graphical interface** on the instrument benchtop allowing to program all the steps that the instrument has to perform. In this step to step guided procedure, some **Report Points** has to be defined at specified times during the experiment, where it is possible to states the volume that has to be injected into the microfluidic system. Once the protocol has been carried out and the sample has been loaded onto the tray, the experiment can start. The experimental protocol is obtained thanks to a well suited **graphical interface** on the instrument benchtop allowing to program all the steps that the instrument has to perform. In this step to step guided procedure, some **Report Points** has to be defined at specified times during the experiment, where it is possible to states the volume that has to be injected into the microfluidic system. Once the protocol has been carried out and the sample has been loaded onto the tray, the experiment can start.
  
-{{ :aree:proteomica:​biacore:​absolute_response.png |}} +{{ :aree:epr:​altre_risorse:​biacore:​absolute_response.png |}}
- +
-Dopo un certo tempo di perfusione del campione, il sistema effettua un lavaggio con il //running buffer// durante il quale si ha il distacco delle molecole di analita che hanno interagito con il ligando. Infine, viene flussato un //tampone rigenerante//​ tipicamente molto salino e basico che consente di staccare tutte le molecole di analita dal ligando, rendendo disponibile il chip ad un nuovo ciclo di legame-distacco.+
  
 After sample injections, molecules of the sample, buffer ions and impurities start interacting with the sensor surface until there is full saturation of the sites, parts of these interactions are not specific, thus just after there is washing out of the fluidic system by running buffer, the detachment of unspecific interactions occurs leaving the real interaction against the baseline. Moreover, according to the experiment, it is also possible to choose two alternative modalities: consecutive injections vs repetitive After sample injections, molecules of the sample, buffer ions and impurities start interacting with the sensor surface until there is full saturation of the sites, parts of these interactions are not specific, thus just after there is washing out of the fluidic system by running buffer, the detachment of unspecific interactions occurs leaving the real interaction against the baseline. Moreover, according to the experiment, it is also possible to choose two alternative modalities: consecutive injections vs repetitive
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 ===== The Sensogram ===== ===== The Sensogram =====
  
-{{ :aree:proteomica:​biacore:​il_sensogramma.png?​400 |}}+{{ :aree:epr:​altre_risorse:​biacore:​il_sensogramma.png?​400 |}}
  
 From the Sensogram it is possible to obtain kinetics information such as association and dissociation by fitting experimental data with specific models as well as thermodynamic information (enthalpy, entropy and Free Gibbs energy) since the device can control the temperature from 4 to 45 °C. \\ From the Sensogram it is possible to obtain kinetics information such as association and dissociation by fitting experimental data with specific models as well as thermodynamic information (enthalpy, entropy and Free Gibbs energy) since the device can control the temperature from 4 to 45 °C. \\
-Amongst the //​[[en:​aree:​proteomica:​biacore:​applicazioni|applications]]//​ are presented some explanatory approaches to catch these parameters.+Amongst the //​[[en:​aree:​epr:​altre_risorse:​biacore:​applicazioni|applications]]//​ are presented some explanatory approaches to catch these parameters.