Hello, 1. Is the extinction coefficient for most dyes such that in a PE-Cy7 tandem the Cy7 will not be able to be excited by the 633 laser because it has not had time to return to its resting state? Theoretically this problem should not have to be addressed, I think. While reading Mario's webstie on tandems he stated that Cy7 in a PECy7 tandem will also be excited by the 633 laser. Even if you can do interlaser compensation, how could you separate the signal from PECy7 and a regular Cy 7 molecule on the same cell. In other words if a tandem is emitting Cy7 fluorescence that was not excited by the PE, how do you separate this signal from a Cy7 alone antibody. 2. We have seen a lot of PE signal from an antibody stained with PECy7. Is this a poorly conjugated fluorochrome? Does this mean that you will have more Cy7 emitting independent of PE excitation? Is this phenomenon common with tandems? Compensation? Seems straightforward if you have another PE antibody that is bright. But what if its not? 3.Are there many of you out there making your own tandems? I guess a lot of these question are addressing the art of setting up multicolor experiments. Apparently its not quite as easy as looking at stokes shifts huh. Thanx in advance, marv
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