FLuorescence methods - (Dec/15/2005 )
again I need your help on fluorescence methods,
Protein 1 is labeled with fluorescein with a 1:1 stechiometry
I would want to observe changes in fluorescence intensity on protein 1 with increasing protein 2 concentrations.
The question is: should I substract intrinsic fluorescence from both protein 1 and protein 2 to obtain net values?
Thanks a lot
intirinsic meaning W and Y? W (H20, pH 7)absorbs at 280nm and emits at 348nm. Y (H20, pH 7)absorbs at 274nm and emits at 303nm. Fluorescein absorbs at 495nm and emits at 520nm. i don't think there should be any overlap to subtract if you are exciting and measuring at the correct wavelength.
and by change do you mean increase(like a FRET method) or quenching (like a FRAP method)?
Yes, intrinsic Trp and Tyr fluorescence.
I do not know "a priori" if the fluorescence will increase or decrease with protein 2.