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I'm currently conducting experiment with fluorescent dye (NBD stearate). This dye binds to hydrophobic part/pockets of proteins and in previous studies, it is usually used to locate organelles (such as mitochondria) where fatty acids commonly binds. In my experiment, I used albumin as my protein and added the dye at optimal condition required for them. We know that albumin also facilitate fatty acid movements in blood stream so it really binds fatty acids.

What I was expecting is that my dye will fluoresce in the presence of hydrophobic pockets of the albumin but when I viewed it even using Fluorescence microscope, no fluorescence at all.

What is that I'm missing here? Why did it not fluoresce?

P.S. Thank you so much for taking time. It's my first time here and I'm wondering if any of you worked with fluorescent dyes/components that had the same problem.