Alexa-488 labeled proteins and receptor binding studies

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Hi all,
    We are using a bacterial protein that is Alexa-488 labeled in order
to determine whether a receptor for this protein exists on responsive
cells (peripheral blood monocytes and THP-1 cells). We have shown some
saturable binding, but the fluorescence is fairly low. The Alexa-488 is
from molecular probes and we've labeled the protein twice, getting 0.8
and 0.4 moles of Alexa dye per mole of protein. We've labeled overnight
at 4C to increase the labeling efficient, but that actually decreased
efficieny. Any one figured out a way to increase the degree of labeling
with this kit?

We would like to determine how many molecules of our Alexa labeled
protein are binding these cells. We dont' need an exact number, but a
rough estimate would be great. Any suggestions?

Thanks,

Slava Epelman

University of Calgary

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