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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.
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.
Aminomethylcoumarin Acetate (AMCA) conjugates absorb light maximally around 350 nm and fluoresce maximally around 450 nm. For fluorescence microscopy, AMCA can be excited with a mercury lamp and observed using a UV filter set. Since blue fluorescence is not well detected by the human eye, AMCA-conjugated secondary antibodies should be used only with the most abundant antigens in multiple-labeling experiments. Ways of improving the visibility of AMCA include dark adapting the eyes, using fluorite instead of glass objectives, avoiding mounting media that absorb UV light (such as plastic-based media), and capturing photographic images with blue-sensitive film or CCD cameras. AMCA fades rapidly in conventional epifluorescence and confocal microscopy, and therefore it should be used with mounting media containing an anti-fading agent such as n-propyl gallate.