Name:

Kd

Category:

Binding

Property:

Dissociation Constant (Kd)

Units:

μM (microMolar)

Measurement Technique:

Fluorescence Polarization/Anisotropy

Buffers:

25 mM HEPES, 1 mM MgCl2, 100 mM NaCl, 0.5 mM tris(2-carboxyethyl)phosphine, and 0.05% Tween 20

Temperature:

25 °C

pH:

7.6

Protein Concentration:

None

Wavelength:

λex = 485 nm, λem = 595 nm

Ionic Strength:

None

Details:

The 2-fold serial dilutions of p97 proteins (maximum 40 μM, purified from bacteria) were mixed with 20 nM BODIPY-FL–ATP (ThermoFisher Scientific) or 10 nM EDA–ADP–ATTO495 (Jena Bioscience) in black low volume 384-well plates. After 30 min, fluorescence anisotropy was recorded on an Envision microplate reader (PerkinElmer Life Sciences) with excitation at 485 nM and emission at 595 nM. Background fluorescence (proteins without added fluorophores) was measured and subtracted from parallel and perpendicular intensities prior to anisotropy calculations. Data were fit in GraphPad Prism 7 using a ligand-binding quadratic equation to obtain Kd values.