Jackson ImmunoResearch

Secondary Antibody Conjugates for
Flow Cytometry

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For flow cytometry we offer three fluorescent proteins (R-PE, APC, and our new PerCP) conjugated to our highly adsorbed secondary antibodies, streptavidin, and purified immunoglobulin controls. The table of Flow Cytometry Products lists all of the antibodies, purified immunoglobulins, and streptavidin to which we conjugate these proteins. Also shown in this table are the same highly adsorbed antibodies and purified immunoglobulins conjugated to Biotin-SP and fluorescent dyes appropriate for flow cytometry (DyLight 488, FITC, and DyLight 649). Note that many antibodies listed elsewhere in tables of Whole IgG and F(ab')₂ Fragments also can be used for flow cytometric applications.

Phycoerythrin (R-PE) and allophycocyanin (APC) are among several kinds of light-harvesting phycobiliproteins found in red, blue-green, and cryptomonad algae. We offer R-phycoerythrin (R-PE), the form found in red macrophytic algae (seaweed). APC is isolated from the blue-green alga Spirulina, and is chemically cross-linked for stability. After phycobiliproteins are conjugated to highly adsorbed secondary antibodies, there is little fluorescence quenching, which results in conjugates of high specific fluorescence compared with conventional fluorophore-antibody conjugates. R-PE and APC can be excited by light over a wide range of the visible spectrum (Figures 1A), are highly water soluble, have relatively low isoelectric points, and lack potentially sticky carbohydrates.

Figure 1. Excitation (A) and emission (B) spectra of DyLight 488/FITC (green), R-PE (red), and PerCP (blue), and excitation (C) and emission (D) spectra of DyLight 649 (brown) and APC (purple). Peak heights were normalized after the spectra were obtained with an M-series spectrofluorometer system from Photon Technology International, Inc.

PerCP is a fluorescent peridinin-chlorophyll protein complex isolated from dinoflagellates. We offer the form found in Dinophyceae sp. with a molecular weight of about 35.5 kDa. It has a broad spectrum of excitation with a main peak at 472 nm, and a long Stokes shift to an emission peak at 677 nm (Figure 1).

PerCP, DyLight 488 (or FITC), and R-PE are excited at 488 nm with an argon laser (Figure 1A), and thus can be used for one- to three-color analyses with single-laser flow cytometers. APC and DyLight 649 are excited at 633 or 635 nm (Figure 1C) to give a fourth color with dual-laser flow cytometers.

It should be noted that the relatively high molecular weights of PerCP, R-PE, and APC may preclude their use in procedures requiring good penetration into cells and tissues. They are predominantly intended for surface labeling of cells for flow cytometry.