F(ab")2 fragment antibodies are generated by pepsin digestion of whole IgG antibodies to remove most of the Fc region while leaving some of the hinge region. F(ab")2 fragments have two antigen-binding Fab portions linked together by disulfide bonds and therefore they are divalent. The average molecular weight is about 110 kDa. They are used for specific applications, such as to avoid binding of secondary antibodies to live cells with Fc receptors or to Protein A or Protein G.
Cyanine Cy™3
Amax: 550Emax: 570nmCy3 is brighter, more photostable, and gives less background than other orange-red fluorescing dye conjugates. Cy3 conjugates can be excited maximally at 550 nm, with peak emission at 570 nm. For fluorescence microscopy, Cy3 can be visualized with traditional tetramethyl rhodamine (TRITC) filter sets, since the excitation and emission spectra are nearly identical to those of TRITC. We recommend Cy3 as a brighter alternative to TRITC. Cy3 can be excited to about 50% of maximum with an argon laser (514 nm or 528 nm lines), or to about 75% of maximum with a helium/neon laser (543 nm line) or mercury lamp (546 nm line). Cy3 has been used with fluorescein for double labeling; however, the use of a narrow band-pass emission filter for fluorescein is recommended to minimize Cy3 fluorescence in the FITC filter set. Cy3 can also be paired with Alexa Fluor® 647 for multiple labeling when using a confocal microscope. However, a better choice for multiple labeling is Rhodamine Red-X because its fluorescence is midway between a green fluorescing dye (like Alexa Fluor® 488) and a far-red-fluorescing dye like Alexa Fluor® 647.
