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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.
Store freeze-dried powder at 2-8°C. When ready to use, rehydrate with indicated volume of d. water and centrifuge if not clear. Product is stable for about 6 weeks at 2-8°C as an undiluted liquid. Prepare working dilution fresh each day. For extended storage after rehydration, add an equal volume of glycerol (ACS grade or better) for a final concentration of 50%, and store at -20°C as a liquid. Note: after the addition of glycerol, the concentration of protein and buffer salts is one-half of the original. Alternatively, aliquot and freeze the product at -70°C or below in the absence of glycerol. Avoid repeated freezing and thawing.
Expiration date: one year from date of rehydration. However, the expiration date may be extended if the product is stored according to the recommendation and the test results are acceptable for its intended use.
1:100 - 1:800 for most applications
Dilution factors are presented in the form of a range because the optimal dilution is a function of many factors, such as antigen density, permeability, etc. The actual dilution used must be determined empirically.
Cy3 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.
This product is for IN VITRO RESEARCH USE ONLY. It is not a medical device and it is not intended for diagnostic or therapeutic purposes.
Cy is a trademark of GE Healthcare Bio-Sciences Ltd. Jackson ImmunoResearch is licensed by GE Healthcare Bio-Sciences Ltd. to manufacture and sell conjugates of Cy2, Cy3, and Cy5 reactive dyes under US Patent Number 5,268,486, and other patents pending.