Considerations before making fluorescence anisotropy measurements
- Homogeneous solution based assay – no separation of bound and unbound material is required.
- Detailed measurements can be made using our dedicated steady state Horiba Jobin Yvon fluorimter.
- Measurements can also be made in 96-well plate for more rapid throughput.
- This is a solution based measurement that is complementary to SPR.
- <mg quantities of material is required.
- Provides equilibrium binding constants.
- The smaller binding partner needs to be labeled with fluorophore. We have seen examples where the attachment of a label can change the binding properties. This needs to be examined.
- Limitations include
- Changes in fluorescence intensity; although these can often be accounted for in data analysis.
- The size of interacting molecules; the smaller molecule needs to be labeled and the theoretical upper size limit to observed changes in anisotropy is 50kDa.
- Rotation of the fluorophore that does not report on the rotation of the molecule it is attached to.
• Changes in fluorescence intensity; although these can often be accounted for in data analysis.
• The size of interacting molecules; the smaller molecule needs to be labeled and the theoretical upper size limit to observed changes in anisotropy is 50kDa.
• Rotation of the fluorophore that does not report on the rotation of the molecule it is attached to.
