I am trying to use a fluorescence probe to study a process involving protein crystals. The protein is Lysozyme, (it contains Tryptophan) and when the protein is in solution I obtain a clear peak in my spectra. The protein was solubilized in NaAc 0.1M and NaCl 0.6M and I excited at 280, 295 and 300 nm and checked emission between 280 and 450 nm . I was able to detect the protein at 25mg/mL of concentration from a drop of 10microliters, directing the probe to the drop with an angle of 45°. Then I did another experiment: I produced my crystals, I washed them (to remove not crystallized protein) and I put them in the same buffer (NaAc 0.1M and NaCl 0.6M). I used a drop of 10microliters again, with the probe in the same position, using same excitation and emission wavelenghts but I was not able to obtain any signal from the protein.
Perhaps the tryptophan fluorescence is quenched in the crystal due to contact with another protein molecule. This would be most likely for a surface exposed tryptophan residue.
I don't know what you are trying to do , but you can conjugate lysozyme to a fluorescent ester such as Alexa 488 from Invitrogen. The reaction is quite easy. As the crystals grow an increase in fluorescence can be followed. Also it not necessary to grow lysozyme crystals in an acetate buffer , you can just use DI water and NaCl (as a precipitant), the crystals are just the same if not better than those grown in the buffer.
See here
Silver, Barry R., and R. Patrick Unwin. "Protein crystals make it big at electrode surfaces." Chemical Communications 41 (2008): 5179-5181.
I agree with the suggestions above, but be careful, most fluorescent spectras are extremely sensitive to their surroundings. A different polarity can change the energy difference between relaxed and excited state, which can shift the fluorescent spectra. Usually the fluorescence is red shifted in aggregated proteins, I guess that could easily happen in your crystal, however you probably don´t see it because the relaxation via fluorescence competes with now much more likely relaxation events like internal conversion and possibly a number of strange effects, so the lifetime and the quantum yield have dropped significantly. These effects can in principle happen to every fluorescent system, however as Barry Silver suggested, Alexa 488 in my experience is in every regard a stable dye. If I were you, I would label my protein with that dye. It hardly bleaches and keeps a constant quantum yield and fluorescent spectra in all the conditions I ever used it.
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