Secondary antibodies for immunodetection, immunofluorescence, and advanced protein analysis
Secondary antibodies are essential tools signal amplification of proteins during immunodetection techniques, in applications like Immunofluorescence (IF), Western blotting (WB), Immunohistochemistry (IHC), and Flow Cytometry (FC). A secondary antibody typically binds with most specificity when the secondary antibody is raised against the species in which the primary antibody was made, thereby enabling enhanced signal amplification and enhanced detection sensitivity.
We offer secondary antibodies in various conjugation formats, including unconjugated, FITC, HRP, AP, biotin, and gold. Our secondary antibodies are supplied in volumes ranging from 100 µL to 7 mL, suitable for both small-scale and high-throughput experiments.
Featured secondary antibody targets
Conjugation options
Secondary antibodies can be conjugated with a variety of labels or tags. We offer multiple conjugation options, including FITC-, HRP-, biotin-, and gold-conjugated secondary antibodies, depending on the experimental technique chosen.
Biotin-conjugated secondary antibodies
Biotin, also known as Vitamin H or B7, can be covalently attached to secondary antibodies, allowing tight binding to streptavidin or avidin.
Alexa Fluor™-conjugated secondary antibodies
High performance dyes ranging from blue to near-infrared. These dyes are significantly brighter than traditional fluorophores, enabling longer imaging sessions and signal capture.
HRP-conjugated secondary antibodies
HRP conjugates are well-suited for Western Blotting and colourimetric detection. HRP is compatible with chemiluminescent and fluorescent substrates.
AP-conjugated secondary antibodies
Alkaline phosphatase (AP)-conjugated secondary antibodies offer high sensitivity and stable signal development. AP produces a strong, long-lasting colorimetric or chemiluminescent signal with low background.
DyLight™-conjugated secondary antibodies
With high photostability and broad spectral range, these conjugates are suitable for long exposure imaging and high signal-to-noise ratios.
Gold-conjugated secondary antibodies
Gold nanoparticle-conjugated antibodies are suitable for electron microscopy and lateral flow assays, offering high-resolution imaging.
Alexa Fluor Secondary Antibody Conjugates
| Name of Dye | Excitation/Emission (nm) | Replaces | Colour |
|---|---|---|---|
| Alexa Fluor 350 | 343/441 | Coumarin (AMCA) | Blue |
| Alexa Fluor 405 | 401/422 | Pacific Blue | Blue |
| Alexa Fluor 488 | 499/520 | FITC, Cy2 | Green |
| Alexa Fluor 532 | 534/553 | Rhodamine Green | Yellow |
| Alexa Fluor 555 | 553/568 | Cy3, TRITC | Orange |
| Alexa Fluor 568 | 579/603 | Rhodamine Red | Red |
| Alexa Fluor 594 | 590/618 | Texas Red | Red |
| Alexa Fluor 633 | 631/650 | Cy5 | Far Red |
| Alexa Fluor 647 | 650/671 | Cy5 | Far Red |
| Alexa Fluor 660 | 663/691 | Cy5.5 | Near-IR |
| Alexa Fluor 680 | 681/704 | IRDye 680, Cy5.5 | Near-IR |
| Alexa Fluor 750 | 752/776 | Cy7 | Near-IR |
| Alexa Fluor 790 | 782/805 | IRDye 800CW | Near-IR |
Dylight Secondary Antibody Conjugates
| DyLight Dye | Excitation (nm) | Applications | Comparable Dyes |
|---|---|---|---|
| DyLight 350 | ~350 | IF, FC | Alexa Fluor 350, AMCA |
| DyLight 405 | ~405 | WB, IF, FC | Alexa Fluor 405, Pacific Blue |
| DyLight 488 | ~488 | WB, IF, FC | Alexa Fluor 488, FITC |
| DyLight 550 | ~550 | WB, IF | Alexa Fluor 546, Cy3 |
| DyLight 594 | ~594 | IF, FC | Alexa Fluor 594, Texas Red |
| DyLight 633 | ~633 | WB, IF | Alexa Fluor 633, Cy5 |
| DyLight 649 | ~649 | IF, FC | Alexa Fluor 647, Cy5.5 |
| DyLight 680 | ~680 | WB, IF, FC | Alexa Fluor 680, IRDye 680 |
| DyLight 800 | ~800 | WB, IF, FC | Alexa Fluor 790, IRDye 800CW |
ABflo Secondary Antibody Conjugates
| ABflo® Dye | Excitation / Emission (nm) | Applications | Comparable Dyes |
|---|---|---|---|
| ABflo® 405 | 405 approx. | IF, ICC, FC | Alexa Fluor 405, DyLight 405 |
| ABflo® 488 | 488 approx. | IF, ICC, FC | Alexa Fluor 488, FITC, DyLight 488 |
| ABflo® 555 | 555 | IF, ICC, FC | Alexa Fluor 555 |
| ABflo® 594 | 594 | IF, ICC, FC | Alexa Fluor 594, Texas Red |
| ABflo® 647 | ≈647 | IF, ICC, FC | Alexa Fluor 647, Cy5, DyLight 649 |
| ABflo® 750 | 750 / 775 | IF, ICC, FC | Alexa Fluor 750, Cy7, APC‑Cy7 |
Rabbit host secondary antibodies
| Specificity (primary antibody target) | Target | Available formats | Research applications |
|---|---|---|---|
| Whole IgG (H+L) | Cat, Chicken, Cow, Dog, Donkey, Duck, Goat, Guinea Pig, Horse, Human, Monkey, Mouse, Pig, Rat, Sheep | Unconjugated, HRP, Alexa Fluor, DyLight, biotin, Cy3, FITC, gold, AMAC | Whole IgG detection, providing versatile detection for multiple assays |
| VHH nanobodies | Llama | HRP, Unconjugated | Specific for nanobody domains; key for camelid antibody studies |
| Anti-IgG Fc-specific Fragment antibody | Human | Alexa Fluor, biotin, FITC, gold, HRP | Binds only to the Fc region of the primary antibody and is useful in avoiding certain types of non-specific binding |
Mouse host secondary antibodies
| Specificity (primary antibody target) | Target | Available formats | Research applications |
|---|---|---|---|
| Whole IgG (H+L) | Chicken, Duck, Human, Monkey, Pig, Rabbit | Unconjugated, HRP, Alexa Fluor, biotin, FITC, Cy3, gold, AMAC | Whole IgG detection, providing versatile detection for multiple assays |
| Light chain-specific antibody | Human, Rabbit | Unconjugated, HRP | Reduces heavy chain noise in WB; suitable for subclass and fragment analysis |
| Fab Fragment-specific antibody | Human | Unconjugated, HRP, Alexa Fluor | Reduces non-specific binding in hybridoma or closely related species assays |
| Anti-IgG Fc-specific Fragment antibody | Human, Rabbit | Unconjugated, Alexa Fluor, AMAC, Cy3, Cy5, Cy5.5, Cy7, FITC | Binds Fc region only; useful to eliminate Fc-mediated background signal |
Goat host secondary antibodies
| Specificity (primary antibody target) | Target | Available formats | Research applications |
|---|---|---|---|
| Whole Ig isotypes (H+L), incl. IgG, IgM, IgA, IgY (chicken) | Chicken, Guinea Pig, Human, Mouse, Pig, Rabbit, Rat | Unconjugated, Alexa Fluor, AMAC, biotin, Cy3, FITC, gold, HRP | Whole IgG detection, providing versatile detection for multiple assays |
| Light chain | Mouse | Gold, HRP, biotin, FITC | Reduces heavy chain noise in WB; subclass and fragment characterisation |
| Fab Fragment | Human | Unconjugated, Alexa Fluor, biotin, FITC, gold, HRP | Reduced non-specific binding to Fc receptors, enabling cleaner staining; useful for blocking endogenous immunoglobulins |
| Anti-IgG Fc-specific Fragment antibody | Human, Rabbit | Unconjugated, AMAC, Alexa Fluor, Cy3, Cy5, Cy5.5, Cy7, FITC | Binds only to the Fc region of the primary antibody and is useful in avoiding certain types of non-specific binding |
Donkey secondary antibodies
| Specificity (primary antibody target) | Target | Available formats | Research applications |
|---|---|---|---|
| Whole IgG (H+L), including highly cross-adsorbed | Goat, Mouse, Rabbit, Rat | Alexa Fluor, biotin, FITC, HRP, ABflo® 488 | Cross-adsorbed secondary antibodies are available, which reduce cross-reactivity and non-specific binding |
Learning and Support
Why use secondary antibodies?
- Signal amplification: Multiple secondary antibodies can bind to a single primary antibody, increasing detection sensitivity — especially useful for low-abundance targets.
- Versatility: Compatible with a wide range of detection systems and experimental formats.
- Multiplexing: Enables the simultaneous detection of multiple targets using different fluorophores.
Choosing the right secondary antibody
- Target species - should be the same as the primary antibody host species.
- Isotype - should be the same isotype as the primary antibody.
- Conjugation - according to your application needs.
We supply affinity-purified secondary antibodies - ensuring high specificity and low background for your experiments.
Isotype and format specificity
Our secondary antibodies are available for all major isotypes (IgG, IgA, IgM, IgD, IgE) and subclasses (e.g., IgG1, IgG2). Our range recognises several primary antibody formats, including:
- Whole IgG: The most common format, suitable for a wide range of applications.
- F(ab')₂ fragments: Ideal for reducing non-specific staining in IHC and IF.
- Fab fragments: Useful in flow cytometry and multiplex immunofluorescence where Fc-mediated binding must be minimised.
Our Guarantee
Our antibodies support demanding applications that require high specificity, producing reproducible results suitable for academic research and publication. They also provide superior batch-to-batch consistency and an unlimited supply, making them ideal for long-term studies. If our products fall below the high standards you expect, we will replace or refund them.
Optimising antibody concentration and dilution
Primary Antibody
- Typical dilution: 1:100 to 1:1,000.
- Optimise based on antigen abundance and tissue type.
Secondary Antibody
- Recommended dilution: 1:500 to 1:2,000 for IF.
- Excessive concentration may lead to high background or non-specific staining.
Troubleshooting Tips
- Use cross-adsorbed secondary antibodies, which have additional manufacturing purification steps to remove off-target species of immunoglobulin (Ig). This enhances specificity and reduces cross-reactivity in immunoassays.
- Include negative controls (no primary antibody) to assess background
- Adjust blocking conditions and washing steps to minimise immunofluorescence background.
For specialised applications
- tdTomato antibody immunofluorescence: Use anti-RFP-compatible secondaries for optimal detection
For Research Use Only - All products are intended for research purposes and are not for use in diagnostic procedures