Immobilization of the Receptor (Integrin α2A-domain) to a Microtiter Plate
2:09
Preparation of a Serial Dilution Row of the Ligand (Rhodocetin)
3:25
Binding of Ligand at Different Concentrations to Immobilized Receptor
5:03
Quantification of Receptor-bound Ligand by ELISA
7:06
Evaluation of the Titration Signals
9:30
Results: Rhodocetin Binds with Lower Affinity to Mutant Integrin α2A-domain
10:58
Conclusion
Transcrição
The overall goal of this titration ELISA is to determine the affinity constant of two binding partners in a reproducible and efficient manner using a novel algorithm. This method can help answer key questions not only in the field of protein ligan
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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.