Secondary Antibody
Tips and technical resources to kickstart your experiments and keep them moving forward, including concentration calculations, centrifuges, sample management, and more.
A secondary antibody binds to another antibody (usually the primary) for detection and amplification. It does not recognize the antigen directly but targets the constant (Fc) region of the primary antibody.
Primary antibodies bind directly to the target antigen, providing specificity. Secondary antibodies recognize and bind to the primary antibody, enabling visualization or signal enhancement in immunoassays.
Properly stored, many secondary antibodies remain stable for months to years. Shelf life is affected by storage temperature, light exposure, and buffer composition.
IgM refers to an antibody isotype, usually present as a primary antibody in immunoassays. Secondary antibodies are not classified by immunoglobulin class but by their detection role and specificity.
IgG is most prevalent in secondary immune responses, owing to its abundance, high specificity, and improved affinity maturation after repeated antigen exposure.
Secondary antibodies allow for signal amplification, better detection sensitivity, and assay flexibility by supporting various conjugation formats for different experimental needs.
Incubation typically ranges from 30 minutes to 2 hours at room temperature, varying by assay type and recommended protocol. Optimization may be needed for the best signal-to-noise results.
Secondary antibodies are designed to bind the Fc region of primary antibodies, acting as detection molecules in assays such as ELISA, Western blot, and immunofluorescence. They enable signal amplification, improving assay sensitivity and allowing broader application options.
Select a secondary antibody that specifically recognizes the host species and isotype of your primary antibody. For example, if your primary is a mouse IgG, choose an anti-mouse IgG secondary with the appropriate conjugation required for your assay.
Conjugated secondary antibodies are linked to a label, such as enzymes (HRP, AP) or fluorophores, enabling detection. Unconjugated secondary antibodies lack these labels and require an additional step for visualization in most assays.
Yes, non-specific binding or cross-reactivity can lead to high background. Proper blocking steps and optimizing antibody dilutions help minimize this issue. Choosing highly validated reagents from AAA Biotech will reduce the risk of undesired signal.
Species cross-reactivity happens if a secondary antibody recognizes Ig from non-target species, leading to off-target binding. Carefully match the host and specificity to your experimental system to avoid such problems.
Common tags include enzymes like horseradish peroxidase (HRP), alkaline phosphatase (AP), and fluorescent dyes such as FITC, Alexa Fluor, and Cy3. These enable either colorimetric or fluorescent signal detection, depending on assay design.
HRP and AP, while both enzyme labels, generate different products and use different detection protocols. They are not always interchangeable; your choice should match the detection needs and substrate compatibility of your assay.
Store secondary antibodies at –20°C or 4°C, depending on manufacturer instructions. Avoid repeated freeze-thaw cycles by aliquoting and keep the product away from light if it’s fluorophore-conjugated.
Direct detection, using a labeled primary antibody, can reduce steps and background, but often at the cost of lower signal amplification. Secondary antibodies are usually chosen when enhanced sensitivity or flexibility is required.
Dilution should be optimized empirically; typical starting dilutions range from 1:1000 to 1:20,000, depending on assay and antibody concentration. Pilot titrations are recommended to minimize background and maximize signal.
It is possible if the secondary antibody specificity matches the application and sample species. However, cross-reactivity and conjugate stability should be considered, and different assay formats may require separate reagents.
Fluorescent secondaries emit light upon excitation and are suited for multiplexing and direct visualization. Enzyme-linked secondaries create a colorimetric or chemiluminescent signal after substrate addition, typically used for quantitative detection.
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