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 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.
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. Bio-Techne offers a wide variety of fluorescent-labeled primary antibodies. Explore our antibodies by type of fluorescent conjugate.
|Fluorescent Conjugate||Excitation Wavelength (nm)||Emission Wavelength (nm)||Emission Color|
|Alexa Fluor® 405||401||421||Blue|
|Alexa Fluor 350||346||442||Blue|
|Alexa Fluor 488||490||525||Green|
|Phycoerythrin (PE)||565 (also 498)||573||Yellow|
|Alexa Fluor 594||590||617||Orange|
|Alexa Fluor 647||653||669||Red|
|Alexa Fluor 700||696||720||Near Infrared|
|Alexa Fluor 750||749||775||Near Infrared|
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.
Products for Signal Amplification
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.
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
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
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.