Thanks for the endorsement of the Alexa Fluro dyes as direct conjugates. They are far easier to conjugate than APC, although APC may give a factor of perhaps 3- to 5-fold in brightness on a molar basis of the antibody due to its much larger size and number of fluorophores. The Alexa Fluor 660 dye has a bit broader absorption than does the Alexa Fluor 633 dye and also a higher extinction coefficient (about double the Alexa Fluor 633 dye). Absorption and fluorescence emission spectra of Alexa Fluor 660 goat anti–mouse IgG (A-21054) in pH 7.2 buffer. [Spectra: 12KB] Fluorescence excitation and emission spectra of Alexa Fluor 633 goat anti–mouse IgG (A-21050) in pH 7.2 buffer. [Spectra: 12KB] However, I expect that the Alexa Fluor 647 dye will probably have the best properties overall of absorbance and higher extinction for direct excitation at 633 nm. Its spectra are quite similar to those of Cy5 but its conjugates tend to be brighter than Cy5 conjugates. Absorption and fluorescence emission spectra of Alexa Fluor 647 goat anti–mouse IgG (A-21235) in pH 7.2 buffer. [Spectra: 12KB] Figure 1.23 Brightness comparison of Molecular Probes' Alexa Fluor 647 goat anti–mouse IgG with Cy5 goat anti–mouse IgG conjugates available from other companies. Human blood was blocked with normal goat serum and incubated with an anti-CD3 mouse monoclonal antibody; cells were washed, resuspended and incubated with either Alexa Fluor 647 or Cy5 goat anti–mouse IgG at equal concentration. Red blood cells were lysed and the samples were analyzed with a flow cytometer equipped with a 633 nm He–Ne laser and a longpass emission filter (>650 nm). [Image] We have also started preparing tandem conjugates of APC with the Alexa Fluor 680, Alexa Fluor 700 and Alexa Fluor 750 dyes, which can presumably be combined with APC to give up to four emissions when excited at 633 nm (probably better with 3, however). Only the Alexa Fluor 680 APC is so far available but when they are all available the spectra will be similar to the figure below. These quantum yields have been normalized and the longer wavelength tandems have lower QYs but the compensation is not too bad. Figure 6.38 Normalized fluorescence emission spectra of (1) allophycocyanin, crosslinked, goat anti–mouse IgG (A-865), (2) Alexa Fluor 680–allophycocyanin goat anti–mouse IgG (A-21000), (3) Alexa Fluor 700–allophycocyanin goat anti–mouse IgG and Alexa Fluor 750 goat anti–mouse IgG. The tandem conjugates permit simultaneous multicolor labeling and detection of up to three targets with excitation by a single excitation source — the 633 nm spectral line of the He–Ne laser. [Image] Presumably one can also do five colors with 488 nm excitation. Figure 6.30 Normalized fluorescence emission spectra of (1) Alexa Fluor 488 goat anti–mouse IgG (A-11001), (2) R-phycoerythrin goat anti–mouse IgG (P-852), (3) Alexa Fluor 610–R-phycoerythrin goat anti–mouse IgG (A-20980) (4) Alexa Fluor 647–R-phycoerythrin goat anti–mouse IgG (A-20990) and (5) Alexa Fluor 680–R-phycoerythrin goat anti–mouse IgG (A-20983). The tandem conjugates permit simultaneous multicolor labeling and detection of up to five targets with excitation by a single excitation source — the 488 nm spectral line of the argon-ion laser. [Image] Howard T Petrie wrote: > Having spent many years conjugating many antibodies to > phycobiliproteins, I can understand why you wouldn't want to do it > yourself (although the results are ALWAYS better). For the last > couple of years, however, we've been using Alexa633 instead, and I > have to say that even using the straight-out-of-the-box protocol > supplied by Molecular Probes, the results are quite good (if you have > enough antibody to spare and want to test a couple of chrome:Ab > ratios the results can be even better). I highly recommend giving it > a try, it takes about one hour of hands-on time and about three hours > in total. > > I should mention that Alexa660 works beautifully as well for flow, in > fact better than 633 (I'd love to hear somebody at either MP or one > of the equipment manufacturers explain why this should be the case; > neither the optics nor the spectra would predict it). In fact after > years as a dye laser loyalist, we now use HeNe exclusively, since we > can simultaneously excite and detect A633 and A660 instead of APC and > TR. Given the utter simplicity of conjugation of the Alexa dyes, > it's worth considering. > > > To All: > > > > I am in need of getting two monoclonal antibodies conjugated to APC. > > If you have had this done by some company/lab and liked the result > >would you please let me know. > > > > thanks > > > > Jim Houston > > -- > > Howard T. Petrie, Ph.D. > Head, Laboratory of Developmental Immunology > Memorial Sloan-Kettering Cancer Center > Box 341, 1275 York Avenue > New York, NY 10021 > phone (212)639-2149 > fax (212)794-4019
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