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Titration ELISA as a Method to Determine the Dissociation Constant of Receptor Ligand Interaction
JoVE 杂志
生物化学
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JoVE 杂志 生物化学
Titration ELISA as a Method to Determine the Dissociation Constant of Receptor Ligand Interaction

Titration ELISA as a Method to Determine the Dissociation Constant of Receptor Ligand Interaction

DOI:
10.3791/57334-v

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12:38 min

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February 15, 2018

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1Institute of Physiological Chemistry and Pathobiochemistry,University of Münster

Chapters

  • 00:04Title
  • 00:45Immobilization of the Receptor (Integrin α2A-domain) to a Microtiter Plate
  • 02:09Preparation of a Serial Dilution Row of the Ligand (Rhodocetin)
  • 03:25Binding of Ligand at Different Concentrations to Immobilized Receptor
  • 05:03Quantification of Receptor-bound Ligand by ELISA
  • 07:06Evaluation of the Titration Signals
  • 09:30Results: Rhodocetin Binds with Lower Affinity to Mutant Integrin α2A-domain
  • 10:58Conclusion

Summary

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A detailed protocol to perform a titration ELISA is described. Moreover, a novel algorithm is presented to evaluate titration ELISAs and to obtain a dissociation constant of binding of a soluble ligand to a microtiter plate-immobilized receptor.

Tags

Titration ELISADissociation ConstantReceptor-ligand InteractionIntegrin Alpha 2 A DomainRhodocetinAffinity ConstantProtein-ligand InteractionReceptor ResearchPharmaceutical ResearchPharmacology

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