Fluorescence Test Chemistry
The binding of 8-anilino-1-naphthalene sulphonate (ANS) to the protein, bovine serum albumin (BSA), provides a convenient test reaction for checking the stopped-flow instrument in emission mode. The fluorescence emission from unbound ANS is low but increases strongly when bound to BSA (quantum yields of 0.004 and 0.75 respectively).
Sample Preparation
| Solution 1:- | 10 -6 M bovine serum albumin in buffer |
| Solute 1: | Bovine Serum Albumin |
| Source: | Sigma A-4378 |
| MW: | 66000 |
| Conc: | 10 -6 M |
Preparation of stock solution (10 -4 M):-
Dissolve 33mg in 5mL of pH7 phosphate buffer
Preparation of working solution (10 -6 M):-
Dilute 0.1mL stock solution in 10mL pH7 phosphate buffer (i.e. 1:100)
Note: The BSA stock solution is the same as is prepared for the Flourescence Polarisation test.
| Solution 2:- | 10 -7 M 8-anilino-1-naphthalene sulphonate |
| Solute 1: | 8-anilino-1-naphthalene sulphonate |
| Source: | Sigma A-1028 |
| MW: | 299 |
| Conc: | 10 -7 M |
Preparation of stock solution (10 -4 M):-
Dissolve 2·99mg in 100mL of water
Preparation of working solution (10 -7 M):-
Dilute 0.1mL stock solution in 100mL water (ie. 1:1000)
Risk Assessment
No special risk. Wash hands as a precaution after handling.
| Measurement Conditions | |
| Syringe Ratio: | 1:1 (both syringes 2.5mL) |
| BSA conc. in cell: | 5x10 -7 M |
| ANS conc. in cell: | 5x10 -8 M |
| Excitation wavelength: | 365nm |
| Slit settings: | 1mm |
| Cut-off filter: | 395nm |
| Acquisition period: | 0.1s |