Jackson Immuno Research Inc.
specializing in secondary antibodies and conjugates

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Whole IgG Affinity-Purified Secondary Antibodies

"We only use secondary antibodies from Jackson ImmunoResearch whenever possible! Customer service is also excellent and the price point for their products is very competitive."

Elizabeth Soberg, University of Washington

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Meet JIR...

Exhibit Schedule

30th Aug - Life Science Exhibits, NYU School of Medicine - New York, NY

13-14th Sep - NIH Research Fall Festival - Bethesda, MD

21-26th Sep - NSH-Nat'l Soc For Histotechnology - St. Louis, MO

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Multiple Labeling

With BV421™ & BV480™

  • Combine with AF488, RR-X, and AF647 for 5-color imaging
  • Compatible with commonly used filter sets
  • Switch nuclear stain from DAPI to DRAQ5 for additional labeling options

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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.

Secondary Antibodies for 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

Whole Molecule or F(ab')2 Secondary Antibody?

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 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
  • Experimental sample
  • Sensitivity required
  • Degree of color separation required

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