Are these proteins at the measurement concentrations you are using, prone to aggregation at the timeframe of 60 minutes?

Did you perform the measurement with an open cuvette?

Are there also chromatic changes in spectra?

Hello Eitan,

ive seen the protein spectra and they don't seem to have changed. The proteins are very stable and I've run a gel analysis to confirm. I've also dialyzed the proteins to the calibrate buffer conditions. It's just that the donor intensity still seems to go up along with the acceptor after 2 hrs. Should I run gel samples along different timepoints ?

Is there any reason to believe your sample is dehydrating throughout time? If it does the concentration can increase hence more fluorescence both for donor  and acceptor.

A gel assay is sometimes not the best way to assess aggregation. Also, aggregates can grow in dialysis. Did you consider performing a ThT fluorescence assay to detect aggregates?

Hello Eitan,

Sorry for the long silence. The protein I work with is a heat shock protein and is very much resistant to aggregation even at temperatures exceeding 65oC. I feel one of the reasons that the fluroscence intensity increases over time may be the fact that the protein stock is a month old. As a control Im purifying a fresh batch of protein. Maybe that will help! If i get similar results, I will be back here posting queries!