I have been measuring my protein solutions' concentrations by diluting them in water 20 fold with a final volume of 100 uL and then measuring the absorbances of these solutions in 96 well plates with plate reader. I don't remember having any problem up until today.
I used 20 mM phosphate buffer instead of water to dilute them today and measured the absorbances at 280 nm repeatedly three times and the absorbance for one of the solutions went up from 0.043 to 0.068 (absorbance of 20 mM phosphate buffer is 0.030 at 280 nM with same volume); I stopped measuring after third one but it would probably go higher as I measured until I hit a plateau.
I measured absorbances of two proteins and only one of them went up that much, other one went up from 0.071 to 0.088; if this were to be concentration dependent I would expect the second solution go even higher but it didn't happen.
I know there may be differences in UV absorbances if protein is folded or unfolded; would it be that dramatic? What is the reason for that increase? I will be grateful for an explanation and a practical solution to the problem.
NOTE: I increased the total volume to 200 uL by simply adding 100 uL of 20 mM phosphate buffer into all wells and the signal increase slowed down a lot; there is still some increase though with 0.001-0.003 increments in each measurement.