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Whole IgG antibodies are isolated as intact molecules from antisera by immunoaffinity chromatography. They have an Fc portion and two antigen binding Fab portions joined together by disulfide bonds and therefore they are divalent. The average molecular weight is reported to be about 160 kDa. The whole IgG form of antibodies is suitable for the majority of immunodetection procedures and is the most cost effective.
Storage and Rehydration: Store freeze-dried solid at 2-8°C. Rehydrate with the indicated volume of dH2O 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.
1:100 - 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.
The adult mouse (C57/Bl6) olfactory bulb was stained for astrocytes (blue), microglia (green), and newborn neuroblasts (red). This picture illustrates where the rostral migratory stream, carrying newborn neuroblasts, enters the olfactory bulb (see dense vimentin staining cutting through the center of the picture). Antibodies used: Astrocytes (Vimentin, AB5733 Millipore and donkey-anti-chicken Cy5, 703-175-155 Jackson ImmunoResearch), Microglia (Iba1, 019-19741 Wako and donkey-anti-rabbit Alexa488, 711-545-152 Jackson ImmunoResearch), and Newborn neuroblasts (marked with BrdU and stained for BrdU, MCA2060T AbD Serotec and donkey-anti-rat Cy3, 712-165-153 Jackson ImmunoResearch).
Carlyn Martina-Mamber, Robin Pronk, Elly M. Hol - Netherlands Institute for Neuroscience
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.