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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.

Page last updated June 12, 2011 @ 7:32 pm