| Assay Method Information | |
| | In Vitro Determination of Compound Activity by HRTF Assay |
| Description: | In the experiment, recombinant Erk1 and Erk2, tagged with a polyhistidine stretch in their N-terminal part were used. To reproduce the interaction between ERK1/2 and Myd88, a peptide corresponding to amino acids 24-40 of Myd88 was used: biotin-Ahx-PLAALNMRVRRRLSLFLNVR with Ahx being aminohexanoic acid as a spacer (biotin-MyD88peptide). This peptide encompassed the interaction motif of Myd88 with the CD domain of ERK1/2. To perform the HTRF assay this peptide was N-terminally labelled with biotin. To detect the interaction between the peptide and Erk1 or Erk2 the following components were used: an antibody directed against the His-tag, labelled with the donor fluorophore (Europium cryptate); The streptavidine protein, labelled with the acceptor XL665 fluorophore directed against the biotin. All the components except Erk1, Erk2 and the peptide were purchased from Cisbio (Condolet, France).Homogeneous Time Resolved Fluorescence (HTRF) assay combines two techniques: Fluorescence Resonance Energy Transfer (FRET) and Time Resolved (TR) measurement. In TR-FRET, a transfer of fluorescence between a donor and an acceptor molecule generates a signal that can then be measured. This energy transfer is only possible when both fluorophores are close enough to each other. Therefore, this method can be used to detect protein-protein interaction. |
| Affinity data for this assay | |
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