Fluorescence Polarisation Test Chemistry - Kinetic Measurement

Phloxine B binds quite well to bovine serum albumin and produces a good polarisation change. The optimal wavelengths when using phloxine are 535nm for excitation and 560nm for emission, however, since glass cut-off filters are used with the fluorescence polarisation accessory, 515nm has been found to be a satisfactory excitation wavelength when used with a 550nm filter.

Sample Preparation

Preparation of stock solution (10 -4 M):-
Dissolve 33mg in 5mL of pH7 phosphate buffer (10mM)

Preparation of working solution (10 -5 M):-
Dilute 1mL stock solution in 10mL pH7 phosphate buffer (ie. 1:10)

Note:   The BSA stock solution is the same as is prepared for the regular flourescence test.

Preparation of stock solution (10 -2 M):-
Dissolve 41.5mg in 5mL of pH7 phosphate buffer

Preparation of working solution (10 -5 M):-
Dilute 0.01mL Stock Solution in 10mL of pH7 phosphate buffer (i.e. 1:1000)

Risk Assessment

Phloxine may irritate the eyes. Wash hands after use.