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.
Storage and Rehydration: Store freeze-dried solid at 2-8°C. Rehydrate with the indicated volume of dH2O (see product specification sheet) and centrifuge if not clear. Prepare working dilution on day of use. Product is stable for about 6 weeks at 2-8°C as an undiluted liquid.
Extended Storage after Rehydration: Aliquot and freeze at -70°C or below. Avoid repeated freezing and thawing. Alternatively, add an equal volume of glycerol (ACS grade or better) for a final concentration of 50%, and store at -20°C as a liquid.
Expiration date: one year from date of rehydration. The expiration date may be extended if test results are acceptable for the intended use.
1:50 - 1:200 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.
RRX (Rhodamine Red-X) conjugates have a peak of excitation at 570 nm and a peak of emission at 590 nm. Although TRITC has been used traditionally with FITC for double labeling, better color separation is achieved by using RRX or Alexa Fluor® 594. Rhodamine Red-X is particularly useful for 3- and 4-color labeling with DyLight 405, Alexa Fluor® 488, and Alexa Fluor® 647 by using a confocal microscope equipped with a 405 nm laser and a krypton/argon laser. Fluorescence from RRX lies about midway between that of Alexa Fluor® 488 and Alexa Fluor® 647, and it shows little overlap with either dye. The krypton-argon laser emits lines at 488 nm, 568 nm, and 647 nm, which are optimal for exciting Alexa Fluor® 488, RRX, and Alexa Fluor® 647, respectively. By adding a 405 nm laser and a 420 nm emission filter, 4-color labeling is possible using DyLight 405-conjugated secondary antibodies from JIR (Figure 5). The separation between all four dyes is perfect for 4-color labeling, and all four dyes are very bright.
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.
Jackson ImmunoResearch Laboratories, Inc. has trademark rights to AffiniPure™.
Rhodamine Red-X is a trademark of Molecular Probes, Inc. Jackson ImmunoResearch is licensed by Molecular Probes, Inc. to manufacture and sell conjugates of Rhodamine Red-X reactive dye.
