For flow cytometry we offer three fluorescent proteins (R-PE, APC, and PerCP) conjugated to many highly adsorbed secondary antibodies, streptavidin, and purified immunoglobulin controls. The table of Secondary Antibodies for Flow Cytometry lists all of the antibodies to which we conjugate these proteins. Also shown in this table are the same highly adsorbed antibodies conjugated to Biotin-SP and fluorescent dyes appropriate for flow cytometry (Alexa Fluor® 488, FITC, and Alexa Fluor® 647). Note that many antibodies listed elsewhere in tables of Whole IgG and F(ab’)2 Fragments also can be used for flow cytometry.
Excitation (left) and emission (right) spectra of Alexa Fluor® 488/FITC (green), R-PE (red), PerCP (blue), Alexa Fluor® 647 (black), and APC (brown). Peak heights were normalized after the spectra were obtained with an M-series spectrofluorometer system from Photon Technology International, Inc.
|Fluorophore||Excitation Peak||Emission Peak (nm)|
|Alexa Fluor® 488||493||519|
|R-Phycoerythrin R-PE||many, 488||580|
|Allophycocyanin APC||many, 650||670|
|Peridinin-Chlorophyll-protein, PerCP||many, 488||675|
Recommended dilution ranges for Flow Cytometry are shown in the table below.
|Unconjugated whole IgG and F(ab’) 2 secondary antibodies||10 – 20 µg / ml|
|Unconjugated Fab secondary antibodies||20 – 40 µg / ml|
|All Alexa Fluor®, DyLight, and Cy3 Conjugates [IgG, F(ab’) 2, Fab]||1:100 – 1:800|
|AMCA, Cy2, FITC, TRITC, RRX, and Texas Red Conjugates [IgG, F(ab’) 2, Fab]||1:50 – 1:200|
|Cy5 Conjugates [IgG, F(ab’) 2, Fab]||1:100 – 1:400|
|Phycoerythrin and Allophycocyanin Conjugates [IgG, F(ab’) 2, Streptavidin]||1:50 – 1:200|
|PerCP Conjugates [IgG, F(ab’) 2, Streptavidin]||1:25 – 1:100|
|Biotin-SP Conjugates [IgG, F(ab’) 2] (using Fluorophore-Conjugated Streptavidin)||1:200 – 1:1,000|
|All Alexa Fluor®-, DyLight 405-, and Cy3-Conjugated Streptavidin||0.5 – 2 µg / ml|
|Cy5-Conjugated Streptavidin||0.5 – 2 µg / ml|
|All Other Fluorophore-Conjugated Streptavidin||2 – 5 µg / ml|