>Who can tell me? What is better to detect a rare molecule: one molecule of >PE or one molecule of APC? > This question simply begs a host of others. The principal one being, against what background? In a vacuum of background fluorescence, PE would be the choice, since it carries more fluors than does APC. However, if you are measuring against a cell, then APC would probably be the winner, since the background autofluorescence is much lower. For that matter, you may find that a tandem dye, like Cy5PE or PE-APC (both commercially available) may give even better sensitivity when measured against cells. This is because the autofluorescence spectrum at the large Stoke's shift exhibited by these molecules is even smaller than that for APC by itself. Then, why are you limiting yourself to one molecule? Why not construct multimers using branched DNA or dextran as the backbone? For that matter, you could use phycobilisomes, which are huge "ex vivo" complexes of phycobiliproteins. Finally, the new "quantum dot" crystals will almost certainly be the winner--for the simple reason that they have a nearly unlimited absorbance, given the right excitation wavelength. It is theoretically possible that you might be able to visualize a single crystal conjugated to an single antibody in a tube--given an x-ray excitation wavelength, probably. But that's still a few years in the future. By the way, you also mention that you are trying to detect a "rare" molecule.... leading me to think that you are using a conjugated antibody. Remember that you will have far greater background from nonspecific binding, plus all the unwanted chemistries that went into an antibody, than would allow for single molecule detection, much less a few dozen. But my point is that you should worry about a host of other factors as well: how good is the chemistry that went into the conjugation, what kind of multimer complexes exist in the conjugate, how much unconjugated antibody is left, how much background does the antibody and even the fluorochrome exhibit, etc. And that doesn't even touch the question of detection: how good are your filters, your laser, your PMT, etc. All of these are critical in determining sensitivity. Bottom line: try it with both, and decide for yourself: be empirical! mr
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