Skip to main content

Using Conjugated Primary Antibodies

Many protein analysis techniques utilize antibodies to detect analytes in complex biological samples. The antibodies used in these immunoassays, such as immunocytochemistry (ICC), immunohistochemistry (IHC), flow cytometry, Western blot, and ELISA, are typically conjugated to enzymatic or fluorescent labels that produce signals to visualize analyte detection. Both primary and secondary antibodies can be conjugated. The decision to use a conjugated primary antibody versus a conjugated secondary antibody is dependent upon whether the protein is being detected by direct or indirect methods.

  • Direct detection is a one-step labeling  procedure. It uses a primary conjugated antibody to identify the target analyte.
  • Indirect detection is a two-step labeling procedure. It utilizes an unconjugated primary antibody to bind to the target protein. A conjugated secondary antibody is then used to detect the bound primary antibody and produce the signal.

Bio-Techne offers an extensive collection of primary conjugated antibodies for your research needs.

Benefits of Using a Primary Conjugated Antibody

Less Background Staining

Using a conjugated primary antibody for direct detection of analytes eliminates any non-specific binding that may occur with secondary antibodies, thereby reducing background signals.

Species Cross-Reactivity Minimized

Using a primary conjugated antibody for direct detection of analytes minimizes species cross-reactivity that may occur with secondary antibodies.

Procedures Simplified

Using a conjugated primary antibody for direct detection of analytes shortens protocols as only one labeling step is required.

Fluorescent Conjugates Available from Bio-Techne

Fluorescent conjugates use fluorochromes to produce a fluorescent signal. They become excited by absorbing a specific wavelength of light. The excited fluorescent dye will then emit light at a longer wavelength. Fluorescent-conjugated primary antibodies can be used for multiplex techniques including immunofluorescence, flow cytometry, and FACS. Additionally, fluorescently labeled primary antibodies are ideal tools for multiplexing. The key is to choose fluorophores that have distinct absorption and emission spectra so that there is no cross-channel fluorescence.

R&D Systems Alexa Fluor 488-conjugated Human Cytokeratin 18 Monoclonal Antibody (Catalog # IC7619) detects Cytokeratin 18 in fixed HeLa cells.
Cytokeratin 18 in fixed HeLa cells.

Bio-Techne offers a wide variety of fluorescent-labeled primary antibodies. Explore our antibodies by type of fluorescent conjugate.

Commonly Used Fluorescent Conjugates for Primary Antibodies

Fluorescent Conjugate Excitation Wavelength (nm) Emission Wavelength (nm) Emission Color
DyLight 405 400  420  Blue 
Alexa Fluor® 405  401  421  Blue 
DyLight 350  353  432  Blue 
Alexa Fluor 350  346  442  Blue 
mFluor™ Violet 450 406  445  Blue 
mFluor Violet 500 410  501  Green 
NorthernLights™ 493  493  514  Green 
DyLight 488  493  518  Green 
Fluorescein (FITC)  498  519 Green
Alexa Fluor 488 490  525  Green 
Alexa Fluor 532 532 554 Yellow
Janelia Fluor® 549 549 571 Yellow
Phycoerythrin (PE) 498, 565  573  Yellow 
NorthernLights 557 557  574  Yellow 
DyLight 550  562  576  Yellow  
mFluor Violet 610 421  613  Orange 
Alexa Fluor 594  590  617  Orange 
DyLight 594 593  618  Orange 
PE/Atto594 498,566  632  Red  
NorthernLights 637 637 658 Red
Allophycocyanin (APC) 650  660  Red 
Janelia Fluor 646  646  664 Red
Alexa Fluor 647 653 669 Red
DyLight 650 652 672 Red
PerCP 488  675  Red 
PE/Cy5.5 498, 566  700  Red  
DyLight 680 692 712 Near Infrared
Alexa Fluor 700 696  720  Near Infrared 
Alexa Fluor 750 749  775  Near Infrared 
PE/Cy7 489, 566 782 Near infrared
APC/Cy7 652  790 Near infrared
Emission Colour Graphic

Enzyme Conjugates Available from Bio-Techne

In addition to fluorescent conjugates, antibodies can also be conjugated to enzymes such as horseradish peroxidase (HRP) and alkaline phosphatase (AP). These enzymes catalyze chromogenic substrates to produce an insoluble, colored precipitate at the site of the antibody-antigen complex. Though the range of colored signals is not as extensive as that with fluorescent conjugates, chromogenic detection of analytes offers several advantages over fluorescence including being amenable to light microscopy and producing a longer lasting signal. Chromogenic detection is ideal for IHC, Western blot, and ELISA.

View Our HRP-Conjugated Primary Antibodies
View Our AP-Conjugated Primary Antibodies
R&D Systems HRP-conjugated Anti-HA Tag Peptide Monoclonal Antibody (Catalog # HAM0601) detects HA-tagged proteins by Western blot.
Detection of HA-tagged proteins by Western blot.

Products for Signal Amplification

Biotin-Conjugated Antibodies

If the expression of the target analyte is low, Biotin-conjugated antibodies can be used, in conjunction with a labeled-Streptavidin molecule, to amplify the detection signal.

Streptavidin Conjugates

Chromogenic- or fluorescent-conjugated Streptavidin is used with Biotin-conjugated antibodies to detect low expressed analytes. A single Streptavidin molecule can bind up to four Biotins, thus amplifying the detection signal.

VisUCyte™ HRP Polymer

The VisUCyte HRP Polymer from R&D Systems, a Bio-Techne brand, is a Biotin-free detection reagent for IHC. It overcomes the problems related to the Avidin-Biotin detection chemistry, thus producing cleaner IHC results.

Additional Antibody Products

Antibody Labeling Kits

The Lightning-Link™ Antibody Labeling Kits from Novus allows you to label primary antibodies in less than 30 seconds hands-on time. Over 40 fluorescent, enzymatic, and biotin labels are available.

Isotype Control Antibodies

We offer isotype control antibodies for negative controls. Our selection includes unconjugated- and conjugated- antibodies for many species and antibody isoforms.

Flow Cytometry Workflow Solutions

We offer a range of products for the entire flow cytometry workflow. Explore our portfolio of flow-validated antibodies and multicolor flow cytometry kits.

Additional Antibody Resources

View of the Immunohistochemistry (IHC) Handbook from BioTechne

Immunohistochemistry (IHC) Handbook

Download Bio-Techne’s IHC Handbook to get information on techniques, protocols, and troubleshooting methods to help with your IHC experiment.

View of the Flow cytometry handbook and a single cell as a background

Flow Cytometry Handbook

Download Bio-Techne’s Flow Cytometry Handbook to get detailed protocols and troubleshooting information for flow cytometry experiments.

western blot handbook cover

Western Blot Handbook

Request this Western Blot eHandbook from Bio-Techne to learn more about the principles of western blot, workflow and protocols, troubleshooting tips, and more.