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. Author manuscript; available in PMC: 2019 Oct 3.
Published in final edited form as: Biochemistry. 2018 Jul 19;57(30):4526–4535. doi: 10.1021/acs.biochem.8b00408

Figure 4.

Figure 4.

Competitive Fluorescence Polarization (FP) binding assays to determine epitope specificity and binding affinity. (a) Schematic representation of the FP assay. Pep-MKK6 labeled with TAMRA dye was first equilibrated with p38α in solution. Subsequently, αp38mb was titrated into the solution of pep-MKK6/p38α complex, thereby displacing pep-MKK6 from its binding site on p38α. (b) FP values were recorded (circular data points) and fit with a competitive binding model (solid line). Loss of polarization with increasing amounts of αp38mb indicates that αp38mb competes for binding to p38α with pep-MKK6 at the same binding site, confirming the epitope-specificity of αp38mb. KD values from three independent experiments were 600±298 nM.