- Table of Contents
Boster offers conjugated secondary antibodies for IHC, ICC/IF, and Western Blotting. These antibodies have been referenced in over 8,000 scientific publications. Best reviews and quality guaranteed.Browse all secondary antibodies
Immunolabeling is critical for many areas of basic or clinical research to detect specific cell or tissue components (antigens) in a sample. This approach can be performed with direct and indirect methods by generating antigen-specific antibodies and fusing tags to antibodies.
For direct immunolabeling, the primary antibody is conjugated to a tag and directly binds to the antigen of interest. The indirect approach involves a conjugated secondary antibody that binds indirectly to the target antigen by binding to the antigen-specific primary antibody.
Secondary antibodies are generated and harvested by immunizing a host animal with an antibody from another species. For example, if the primary antibody is a rabbit polyclonal antibody, an anti-rabbit secondary antibody is raised in a different host species such as goat. The produced secondary antibody's specificity is dependent on the immunizing antibody's characteristics, such as species, subclass, fragment, etc.
For a successful experiment, it is important to have a good understanding of the primary and secondary antibodies. Choosing a suitable approach and/or antibodies will better serve the purpose of the experiment while saving money, time, and precious sample. Although monoclonal or polyclonal primary and secondary antibodies can be used for experiments, there are some differences between the primary and secondary antibodies that should be considered.
Primary antibodies bind directly to the antigen by recognizing a specific area/domain (epitope) of the antigen, whereas secondary antibodies bind to the primary antibody—not directly to the antigen. While primary antibodies are necessary for every immunoassay, secondary antibodies are not always required—depending on the experimental method (direct/indirect).
As previously mentioned, secondary antibodies must be made in a species different from those of the primary antibody or the specimen to minimize non-specific binding that leads to false positives and high background noise. Secondary antibodies may also require an extra purification process (pre-adsorption) with a column matrix during the immunoassay to remove non-specific antibodies and increase specificity.
With its single labeling step, the direct method offers a shorter assay time with a simpler workflow. Since it minimizes species cross-reactivity and non-specific binding, the direct method is best used for specific targeting if multiple antibodies are raised in the same species. However, this method demands an abundant supply of expensive conjugated antibodies—with few color selections and limited range of reporter molecules available—and is much less practical than the indirect method.
Although the indirect method requires additional steps, time, and added complexity, it still offers several advantages over the direct method. More than one secondary antibody can specifically bind to different parts of the same primary antibody—increasing the versatility, antigen signal detection, and amplification. The indirect method also contributes to the detection, sorting, and purification of target antigens—providing higher degrees of specificity and sensitivity. Commercially available conjugated secondary antibodies are relatively inexpensive and available in a wider spectrum of colors compared to conjugated primary antibodies—with increased access to several different probes.
By using primary antibodies as a ‘bridge’ to bind with the target antigen, secondary antibodies reduce the possibility of the reporter molecule compromising the binding capability to the antigen epitope. If the target antigen is expressed at a low concentration, using secondary antibodies will allow for multiplexing or multi-labeling across applications (e.g. immunofluorescence, western blot) to validate target antigen detection.Browse all secondary antibodies
Selecting the right secondary antibody is essential for the successful detection of the target antigen. Based on the application, primary antibody, and experimental design, some factors should be considered when selecting a suitable secondary antibody, such as:
In general, whole secondary antibodies containing both heavy (H) and light (L) chains of the Ig are more widely used. Whole secondary antibodies will give higher signal due to their stronger binding to variable regions—with sufficient regions for the attachment of enzymes and dyes. However, whole antibodies can increase cross-reactivity and lower specificity, so it may sometimes be preferable to use a fragment to eliminate non-specific binding.
There are two types of fragments, namely F(ab) and F(ab’)2. With its single binding site, F(ab) fragments (MW=~50kDa) are useful in blocking background noises caused by primary antibodies and endogenous Ig in the sample material. F(ab’)2 fragments are bigger (MW=~110 kDa) and may penetrate tissues/cells more easily. This trait—coupled with their strong divalent bonding in the variable regions—ensures the secondary does not bind to the cell surface.
For detection and visualization of the presence of the target protein, the antibodies are conjugated with probes/markers. There are different types of conjugates, depending on the application and detection technology (colorimetric, chemiluminescent, or fluorescent). Below is a description of the most commonly used secondary antibody conjugates:
Fluorophores emit light in the visual range when excited by light at a particular wavelength—which is then detected by the fluorescent microscope. There are several types of fluorophore-conjugated secondary antibodies available (Alexa Fluor®, DyLight®, IRDye®, FITC, etc.), all with their own excitation and emission characteristics.
Enzymes such as horseradish peroxidase (HRP) and alkaline phosphatase (AP) are capable of converting soluble, colorless substrates into a water-insoluble colored precipitate, which allows visualization with colorimetric or chemiluminescent detection (western blot, immunochemistry).
Biotin is a small vitamin molecule that can be easily modified and/or attached to proteins, antibodies, and other biomolecular probes of interest (e.g. avidin or streptavidin protein). The signal amplification following the interaction between biotin with enzyme- or fluorochrome-labeled secondary antibodies makes it suitable for detecting proteins expressed at low levels.
Colloidal gold conjugates are primarily suitable for immunoassays using an electron microscope. However, gold conjugated secondary antibodies are also used for applications such as flow cytometry, bio-imaging, and lateral flow. The gold nanoparticles can be provided in varying sizes (e.g. 1.4nm, 5nm, 10nm, 40nm).
We provide several different conjugates for secondary antibodies - HRP, biotin, FITC, DyLight®, colloidal gold, etc. - as well as unconjugated secondary antibodies in our catalog
Most secondary antibodies are used in low concentrations between 1 and 10 μg/mL since excessive amount of secondary antibodies will result in high background noise from too much non-specific binding. For optimal performance with minimal background interference, a good starting concentration for a typical secondary antibody in that concentration range would be a dilution of 1:1,000. However, if the staining is extremely bright or the reading shows too much background, higher dilutions from 1:2,000 to 1:20,000 should be considered.
Incubation of the secondary antibodies for 1 hour (37°C) or 2 hours at room temperature should be sufficient. For a stronger signal, the incubation should be performed overnight with agitation at 4°C.
It should be noted that the dilution ratio and incubation time for the secondary antibodies will vary for each application. The end user will need to determine the optimal dilution ratio and incubation time for each application based on the experimental conditions, such as the antigen/antibody concentrations, pH, temperature, and buffer constituents. Optimization of the antibody dilution can be done by performing titration experiments. The table below outlines the recommended secondary antibody types for each application.
|Application||Secondary Antibody Types|
Browse all the detection kits and secondary antibodies below.
|Specificity||Secondary Antibody||Detection Kits With Secondary Antibody|
|Avidin||DyLight®488, HRP, TRITC, FITC, Cy3|
|Donkey Anti Mouse IgG||HRP, DyLight®594, DyLight®550, DyLight®488, Biotin, Biotin||HRP, HRP|
|Donkey Anti Mouse IgM μ Chain||Biotin|
|Donkey Anti Rabbit IgG||HRP, DyLight®594, DyLight®550, Biotin||HRP|
|Donkey Anti Rat IgG||Biotin||HRP|
|Goat Anti Human IgA||FITC, HRP|
|Goat Anti Human IgM||HRP, FITC, Biotin|
|Goat Anti Mouse IgG||HRP, Biotin, DyLight®488, FITC, TRITC, DyLight®594, Unconjugated, FITC, Cy3, 5 nm Colloidal Gold, 10 nm Colloidal Gold||HRP, HRP, Alkaline Phosphatase, HRP, HRP, HRP, Alkaline Phosphatase, FITC, Cy3, DyLight®488, FITC + POD, HRP, HRP|
|Goat Anti Mouse IgM||HRP, Biotin||HRP, Alkaline Phosphatase, FITC, Cy3, DyLight®488, FITC + POD|
|Goat Anti Rabbit IgG||TRITC, Biotin, DyLight®488, FITC, HRP, DyLight®594, Unconjugated, Cy3, 5 nm Colloidal Gold, 10 nm Colloidal Gold||HRP, HRP, Alkaline Phosphatase, HRP, HRP, Alkaline Phosphatase, FITC, Cy3, DyLight®488, FITC + POD, HRP, HRP|
|Mouse Anti Human IgG||FITC, Biotin|
|Mouse Anti Rabbit IgG||Biotin||HRP|
|Protein A||Biotin, FITC, HRP, 5 nm Colloidal Gold, 10 nm Colloidal Gold|
|Protein G||Biotin, FITC, Peroxidase, 5 nm Colloidal Gold, 10 nm Colloidal Gold|
|Rabbit Anti Avidin||HRP|
|Rabbit Anti goat IgG||Biotin, FITC, HRP, TRITC, Unconjugated, Cy3||HRP, HRP, Alkaline Phosphatase, FITC, Cy3, DyLight®488, FITC + POD, HRP, HRP|
|Rabbit Anti Human IgG||FITC, HRP, TRITC, Unconjugated, Biotin, Cy3||HRP, HRP, Alkaline Phosphatase, FITC, Cy3, DyLight®488, FITC + POD|
|Rabbit Anti Mouse IgG||Unconjugated|
|Rabbit Anti Rat IgG||Biotin, FITC, HRP, Unconjugated||HRP, HRP, Alkaline Phosphatase, FITC, Cy3, DyLight®488, FITC + POD|
|Donkey Anti Goat IgG||HRP|
|Donkey Anti Mouse IgM||HRP|