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"I have used a wide variety of secondaries and Jackson ImmunoResearch has consistently been the best. The fluorophores are bright and stable and their selective (x reactivity removed) secondaries have always shown species specificity in multiple labeling."
Janet Duerr, Ohio University
Flow cytometry is a powerful technique for measuring and analyzing the physical characteristics of single particles in solution as they travel past a beam of light. Properties such as relative size and fluorescence can be measured, and fluorescently tagged antibodies enable cells to be interrogated for multiple surface proteins and molecular dynamics. Isotype controls are used for experiment validation and analysis of results.
Flow cytometry can be performed directly using conjugated primary antibodies, or indirectly, by using a conjugated secondary antibody to bind an unconjugated primary. Indirect flow cytometry allows the choice of a wide range of probe molecules, enabling the user to match the desired probe with any primary antibody. Secondary antibody conjugates can improve a flow cytometry experiment by preserving the active site of the primary antibody, and by signal amplification.
Direct (A) and indirect (B) flow cytometry.
The format of
F(ab') 2 fragments are generated by proteolysis of the whole IgG to yield a divalent fragment containing two Fab arms and no Fc domain. When used to stain tissue or cells, the F(ab')2 secondary antibodies can help to avoid background caused by off-target binding. The absence of an Fc region prevents F(ab')2 antibodies from being captured by Fc receptors expressed on cell surfaces.
Please note that if a primary antibody is trapped by an Fc receptor, the F(ab')2 secondary antibody will detect the off-target binding, so blocking is critical.
FabuLight™ secondary antibodies are created by papain digestion of IgG, followed by removal of Fc fragments. These monovalent Fab fragments are specific for the Fc region of primary antibodies, so they don’t interact with the primary’s antigen-binding region. Conjugated FabuLights are convenient for labeling primary antibodies prior to incubation with an experimental sample, saving incubation and wash steps. Like F(ab') 2 fragments, these Fab fragments can minimize background staining due to Fc receptor binding.
The choice of fluorescent dye conjugate depends on a number of experimental variables.
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 and purified immunoglobulins 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.
|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|
Jackson ImmunoResearch offers Biotin-SP conjugated secondary antibodies in both whole IgG and F(ab') 2 format. Biotin-SP conjugates require the use of fluorescently labeled streptavidin for visualization.