James Marvin wrote: >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. Cy7 has a pretty low extinction coefficient at 633 nm; if there is any excitation from the 633 nm laser, there may be an energy transfer effect. Cy5 and Cy5.5 will be directly excited by 633 or 635 nm lasers. But see below. >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? The spectral overlap between PE and Cy7 is not very large, meaning that, in a pure PE-Cy7 tandem, energy transfer between PE and Cy7 will be relatively inefficient, resulting in substantial emission from PE. I believe that at least some PE-Cy7 tandems have another dye attached that accepts energy from PE and transfers it to Cy7 with enough efficiency to minimize PE emission (and maximize Cy7 emission) from the tandem. Matrix compensation should be able to deal with donor emission from tandems (i.e., PE emission from PE tandems, APC emission from APC tandems, etc.). -Howard
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