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Common Antibody Conjugates for Research
Common Antibody Conjugates for Research
Antibody conjugates are essential tools in biological research, offering both specificity and sensitivity for detecting and quantifying proteins, cells, and other molecules. Below, we explore the most common types of antibody conjugates, their examples, applications, and popularity in research.
What is antibody conjugation?
Antibody conjugation is the process of chemically linking an antibody to another molecule, such as a fluorescent dye, enzyme, biotin, or nanoparticle. This process enhances the antibody’s ability to detect specific targets by enabling visualization or measurement in various assays. Conjugated antibodies are widely used in research for applications like flow cytometry, ELISA, and immunofluorescence, where they facilitate the detection and analysis of specific proteins or cells in complex samples.
Common types of antibody conjugates
Fluorophore Conjugates
Among the most commonly used are fluorophore conjugates, which include dyes like fluorescein isothiocyanate (FITC), cyanine dyes, DyLight® dyes, allophycocyanin (APC), phycoerythrin (PE), R-phycoerythrin (R-PE), and iFluor® dyes.
- Fluorescein isothiocyanate (FITC): FITC is a green fluorescent dye commonly used in flow cytometry and immunofluorescence microscopy.
- Cyanine Dyes (e.g., Cy3, Cy5): Cyanine dyes are used for multiplexing due to their distinct spectral properties.
- DyLight® Dyes: The DyLight® dyes are a series of high-performance dyes known for their photostability, brightness, and versatility. Popularly used DyLight® dyes include DyLight® 488, 550, 594, 650, and 800 are popularly used in applications such as flow cytometry, immunofluorescence microscopy, and Western blotting.
- Allophycocyanin (APC): APC is a red fluorescent protein used in flow cytometry for its high quantum yield.
- Phycoerythrin (PE): PE is a general term for phycoerythrin proteins derived from various algae species. It is widely used in flow cytometry and fluorescence microscopy due to its bright fluorescence.
- R-phycoerythrin (R-PE): R-PE, derived specifically from red algae, is a highly bright red-orange fluorescent protein used in flow cytometry and other fluorescent applications. It offers even greater brightness due to its multiple chromophores. This makes R-PE ideal for applications requiring high sensitivity and resolution, such as multicolor flow cytometry.
- iFluor® Dyes: iFluor® dyes, including iFluor® 488, 555, 594, 647, and 750, are designed for superior brightness and photostability, making them excellent choices for advanced fluorescence imaging techniques and multicolor applications.
Fluorophore-conjugated antibodies are widely used in:
- Flow Cytometry: For analyzing cell populations by measuring fluorescence intensity.
- Immunofluorescence: For visualizing protein localization in cells or tissue sections.
- Confocal Microscopy: For high-resolution imaging of fluorescently labeled samples.
Below, we have provided a table comparing key characteristics and uses of some of the most common fluorophore conjugates in research.
| Fluorophore | Color | Max Excitation (nm) | Max Emission (nm) | Extinction Coefficient (M⁻¹cm⁻¹) | Advantages | Applications |
|---|---|---|---|---|---|---|
| FITC | Green | 495 | 519 | 70,000 | Bright, photostable, common filter sets | Flow cytometry, immunofluorescence, microscopy |
| Cy3 | Orange | 552 | 570 | 150,000 | Bright, used in multiplexing | Flow cytometry, immunofluorescence, FISH |
| Cy5 | Red | 650 | 670 | 250,000 | Near-infrared, high sensitivity | Flow cytometry, imaging, FRET |
| DyLight® 488 | Green | 493 | 518 | 70,000 | Bright, photostable | Flow cytometry, immunofluorescence, microscopy |
| DyLight® 550 | Orange | 562 | 576 | 150,000 | High brightness, photostable | Western blotting, fluorescence microscopy, flow cytometry |
| DyLight® 594 | Red | 593 | 618 | 115,000 | Bright, minimal spectral overlap | Multicolor fluorescence imaging, flow cytometry |
| DyLight® 650 | Far-red | 652 | 672 | 250,000 | Near-infrared, reduced background | Flow cytometry, fluorescence imaging |
| DyLight® 800 | Near-IR | 783 | 800 | 270,000 | Near-infrared, minimal autofluorescence | In vivo imaging, Western blotting, NIR fluorescence imaging |
| iFluor® 488 | Green | 491 | 516 | 70,000 | Bright, photostable, FITC alternative | Flow cytometry, immunofluorescence, confocal microscopy |
| iFluor® 555 | Orange | 555 | 565 | 150,000 | High brightness, photostable | Fluorescence microscopy, flow cytometry, multicolor applications |
| iFluor® 594 | Red | 590 | 615 | 115,000 | Bright, minimal spectral overlap | Multicolor fluorescence imaging, flow cytometry |
| iFluor® 647 | Far-red | 650 | 665 | 250,000 | High brightness, photostable | Flow cytometry, fluorescence imaging, super-resolution microscopy |
| iFluor® 750 | Near-IR | 755 | 779 | 270,000 | Near-infrared, minimal autofluorescence | In vivo imaging, NIR fluorescence imaging |
| APC | Red | 650 | 660 | 700,000 | High quantum yield, photostable | Flow cytometry, imaging |
| PE | Orange | 480-565 | 575-590 | 1,960,000 | High brightness, quantum yield | Flow cytometry, fluorescence microscopy |
| R-PE | Red-orange | 488, 546, 565 | 575-585 | 1,960,000 | Extremely bright, multiple chromophores | Flow cytometry, high sensitivity applications |
Fluorophore conjugates are very popular due to their versatility, high sensitivity, and the variety of available dyes that allow multiplexing. When searching for primary antibodies and secondary antibodies at Boster, you’ll be able to select from a range of conjugation options, such as Cy3, DyLight® dyes, FITC, APC, PE, or iFluor® dyes. You can also request custom antibody conjugation with our antibody conjugation service, which offers more conjugate labels.
Enzyme Conjugates
Enzyme conjugates, such as those linked to horseradish peroxidase (HRP) and alkaline phosphatase (AP), are also commonly used in research. These conjugates are crucial in assays like ELISA, WB, and IHC.
- Horseradish Peroxidase (HRP): HRP is an enzyme that catalyzes the oxidation of substrates, producing a detectable signal. The conjugate is regularly used in ELISA and Western blotting. HRP is particularly favored for its high signal-to-noise ratio, making it a staple in laboratory assays.
- Alkaline Phosphatase (AP): AP is an enzyme that hydrolyzes phosphate groups, and this conjugate can be utilized in ELISA, Western blotting, and immunohistochemistry.
Enzyme-conjugated antibodies are used in:
- ELISA (Enzyme-Linked Immunosorbent Assay): For quantitative measurement of proteins in samples.
- Western Blotting: For protein detection after gel electrophoresis.
- Immunohistochemistry: For detecting antigens in tissue sections using colorimetric reactions.
Enzyme conjugates are highly popular in routine laboratory assays due to their robustness and ease of use. At Boster Bio, you can find primary antibodies and secondary antibodies conjugated to HRP, AP, and more. In addition, you can select specific conjugates for your antibodies with our custom antibody conjugation service.
Biotin Conjugates
Biotin, a vitamin that can be easily bound by streptavidin, has proven to be another essential antibody conjugate in research. It provides significant advantages due to its amplification capabilities. Biotin-labeled antibodies, often paired with streptavidin-HRP or AP, are used by researchers in ELISA, Western blotting, and immunohistochemistry.
In research, biotin-conjugated antibodies are frequently used in:
- ELISA and Western Blotting: Paired with streptavidin-HRP or AP for enhanced sensitivity.
- Affinity Purification: For isolating proteins or complexes from samples.
- Immunohistochemistry: As a versatile tool with amplification steps.
Biotin conjugates are widely used due to their ability to provide amplification for applications that require high sensitivity. Boster Bio's catalog contains biotin-conjugated primary antibodies and secondary antibodies, and additional conjugate options. You can also learn more about our custom antibody conjugation service and book a meeting with us to discuss your project, so we can better serve your research needs. Submit an inquiry today!
Metal Conjugates
Metal conjugates, including lanthanide-chelated antibodies (e.g., Europium, Terbium) and metal isotope-tagged antibodies for mass cytometry (CyTOF), are gaining traction in advanced applications.
- Lanthanide-chelated antibodies (e.g., Europium, Terbium): These antibodies are used in time-resolved fluorescence assays.
- Metal Isotope-tagged antibodies for CyTOF (Mass Cytometry): Metal isotope-tagged antibodies allow high-dimensional analysis of cell populations.
Metal-conjugated antibodies are used in:
- Mass Cytometry (CyTOF): For high-dimensional analysis of cell populations, offering detailed phenotyping with minimal signal overlap.
- Multiplexed Immunoassays: Where lanthanides enable time-resolved fluorescence.
Growing popularity of metal conjugates, especially in advanced applications like CyTOF, reflects their capability to provide comprehensive cellular analysis.
Quantum Dot Conjugates
Quantum dot conjugates are semiconductor nanoparticles, including Qdot 525 and Qdot 655, known for their unique optical properties.
Quantum dot-conjugated antibodies are used in:
- Fluorescence Microscopy: For long-term imaging with high photostability.
- Multiplexed Imaging: Due to their broad excitation and narrow emission spectra.
Although less common than traditional fluorophores, quantum dots (Qdots) are increasingly popular in imaging applications for their photostability and distinct spectral properties.
Gold Nanoparticle Conjugates
Gold nanoparticles (AuNPs) are widely employed in various diagnostics, biosensing, and imaging applications.
Gold nanoparticle-conjugated antibodies are used in:
- Lateral Flow Assays: For rapid point-of-care testing (e.g., pregnancy tests).
- Electron Microscopy: For enhanced contrast in imaging.
- Biosensors: For detecting various analytes with stability and high sensitivity.
Gold nanoparticle-conjugated antibodies are quite popular in diagnostics and increasingly in biosensing applications due to their practical utility and ease of detection.
Conclusion
Antibody conjugates play a vital role in modern research, with each type offering distinct advantages. Fluorophore and enzyme conjugates remain staples due to their broad applications and established protocols. Biotin conjugates are favored for applications requiring high sensitivity, while metal conjugates offer advanced analysis capabilities. Quantum dots and gold nanoparticles, though more specialized, are expanding in use as techniques and technologies improve. Selecting the appropriate conjugate type based on your research needs can significantly enhance your experimental outcomes and data quality.
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