Jackson Immuno Research Inc.
specializing in secondary antibodies and conjugates

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Whole IgG Affinity-Purified Secondary Antibodies

"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

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Secondary Antibodies

For VHH Discovery

Anti-Alpaca IgG, Subclass and VHH domain secondaries
  • Ideally time PBMC harvest
  • Enhance detection of VHH antibodies
  • Test for VHH expression and binding activity

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Secondary Antibodies for Flow Cytometry

Secondary Antibodies for Flow Cytometry

Introduction to flow cytometry

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.

Indirect flow cytometry

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 and Indirect Flow Cytometry Direct (A) and indirect (B) flow cytometry.

Secondary antibody format for flow cytometry

The format of secondary antibody can impact the success of an experiment. In addition to whole molecule IgG, Jackson ImmunoResearch offers fragments of secondary antibodies.

F(ab')2 Secondary Antibody

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.

Browse F(ab')2 Secondary Antibodies

FabuLight™ - Fc specific Fab fragments

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.

Read More about FabuLight™

Fluorescent conjugates for flow cytometry

The choice of fluorescent dye conjugate depends on a number of experimental variables.

  • Instrument capabilities. Consider excitation capabilities (excitation wavelengths or laser colors),and which filters are available.
  • Experimental sample. It may be necessary to consider autofluorescence or the expressionof recombinant fluorescently tagged proteins, which may preclude using fluorophores withspectral overlap.
  • Sensitivity required. Several fluorophores may have similar excitation and emission spectra, butdifferences in inherent brightness can result in one fluorophore showing a larger population shift.For example, Alexa Fluor® 488 is brighter than FITC.
  • Degree of color separation required. For multiple labeling, the dye panel choices will beconstrained by the equipment available. To achieve good color separation it is important to lookat the emission overlap of the fluorophores in the dye panel. Panel developing tools are availableonline which can help build dye panels specific to any instrument.

Fluorescent Protein Conjugates

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.

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.

Browse fluorescent proteins conjugated to secondary antibodies

Fluorophore Excitation Peak Emission Peak (nm)
Alexa Fluor® 488 493 519
FITC 492 520
R-Phycoerythrin R-PE many, 488 580
Allophycocyanin APC many, 650 670
Peridinin-Chlorophyll-protein, PerCP many, 488 675

Using JIR Secondary Antibodies for Flow Cytometry

Recommended dilution ranges for Flow Cytometry are shown in the table below.

Product Flow Cytometry
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

Biotin-SP conjugates for flow cytometry

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.

Browse Streptavidin conjugates

Learn more about Biotin-SP

Learn more about signal amplification