Which groups are on your target?
Consider NHS for primary amines and Maleimide for free thiols. For click chemistry, confirm the reaction partners first.
AF514 fluorescent dyes
Green emission, with 5 reactive dyes and 3 bioconjugates. Excitation near 514 nm and collection near 542 nm; find labeling methods and documents for the target handle.
517nm
542nm
8
Optical values refer to AF514 NHS.
AF514 NHS spectral referenceGreen
AF514 product selection
Find AF514 dyes and conjugates by reactive group or recognition ligand.
Which recognition ligand do you need?
Compare Annexin V, WGA, CTB; check the sample and conditions.
| Product / catalog no. | Reactive form / ligand | Targets and applications | Documents |
|---|---|---|---|
| AF514 NHS EsterQH-10-1005 | NHS | Antibodies, proteins and other molecules with accessible primary amines | View product → |
| AF514 MaleimideQH-10-1005(Maleimide) | Maleimide | Targets with accessible free thiols | View product → |
| AF514 AzideQH-10-1005(Azide) | Azide | Targets with a compatible alkyne; confirm the click reaction system | View product → |
| AF514-PEG4-DBCOQH-10-1005(DBCO PEG4) | PEG4-DBCO | Azide coupling with a PEG4 spacer | View product → |
| AF514-PEG12-DBCOQH-10-1005(DBCO PEG12) | PEG12-DBCO | Azide coupling; evaluate a PEG12 spacer | View product → |
| AF514-Annexin VQH-10-1005(Annexin V) | Annexin V | Recognizes exposed PS; binding requires Ca²⁺ | View product → |
| AF514 WGAQH-10-1005(WGA) | WGA | Recognizes glycans containing GlcNAc or sialic acid | View product → |
| AF514 CTBQH-10-1005(CTB) | CTB | Recognizes GM1; consider binding and uptake conditions | View product → |
Showing 8 / 8 AF514 products
Spectra and detection conditions
AF514 NHS optical properties
- Reference product
- AF514 NHS · QH-10-1005
- Excitation peak
- 517 nm
- Emission peak
- 542 nm
- Molar extinction coefficient
- 80,000 M⁻¹ cm⁻¹
Values refer to this product’s specifications. Consult measurement records for solvent, pH and conjugation state.
Full product specifications →Excitation near 514 nm and collection near 542 nm
Begin evaluation with a 514 nm source or an appropriate excitation band. With a 488 nm source, measure signal and background under those conditions rather than carrying over AF488 exposure or gain.
A 540/30 bandpass nominally spans 525–555 nm and includes the 542 nm emission peak. Collected signal depends on the full emission spectrum, filter transmission and detector response; this is a passband example. When comparing AF488 and AF514, assess how the same green detection window collects each signal.
Read the detection guide →Multicolor combinations and controls
AF514 emission lies between AF488 and AF532, so nearby emission windows can collect neighboring labels. For samples containing GFP or FITC, use single-stain and unstained controls and inspect separation after compensation or unmixing.
Multicolor detection considerations →Match the workflow to the target handle
Bioconjugates
For established recognition ligands, review the corresponding conjugates and their product-specific formulation, dose and storage conditions.
| Product | Recognition target and conditions |
|---|---|
| AF514-Annexin V | Phosphatidylserine; binding is Ca²⁺ dependent. |
| AF514 WGA | GlcNAc- or sialic-acid-containing glycans; consider sample preparation and intracellular signal. |
| AF514 CTB | GM1 ganglioside; confirm the application and treatment conditions. |
References
References on reaction chemistry, assay design and result interpretation.
Labeling a protein with fluorophores using NHS ester derivitization
Nanda JS, Lorsch JR. · Methods in Enzymology · 536:87–94 · 2014
Uses FAM ester labeling of a protein to explain primary-amine chemistry and labeling-efficiency assessment. It informs NHS workflow design; input and reaction conditions depend on the actual dye and sample.
Fluorescent-labeled antibodies: Balancing functionality and degree of labeling
Vira S, Mekhedov E, Humphrey G, Blank PS. · Analytical Biochemistry · 402(2):146–150 · 2010
Uses FITC-labeled anti-HA antibody to assess changes in labeling density and antigen binding. It informs input-series design and post-purification functional testing; DOL alone is insufficient for selecting a conjugate.
Guidelines for the use of flow cytometry and cell sorting in immunological studies (third edition)
Cossarizza A, Chang HD, Radbruch A, et al. · European Journal of Immunology · 51(12):2708–3145 · 2021
Provides methodological guidance on detection settings, controls and experimental records for downstream conjugate evaluation.
Technical guides
AF514 · Labeling & detectionAF514 spectra and detection channelsAF514 NHS has 517/542 nm peaks. Check the excitation source, emission window and detector response separately.
Product peaks and excitation source
AF514 NHS has an excitation peak of 517 nm and an emission peak of 542 nm. Check the product name, source wavelength and collection window separately. Begin evaluation with a 514 nm source or an appropriate excitation band. With a 488 nm source, measure signal and background under those conditions rather than carrying over AF488 exposure or gain.
Emission window and optical path
A 540/30 bandpass nominally spans 525–555 nm and includes the 542 nm emission peak. Collected signal depends on the full emission spectrum, filter transmission and detector response; this is a passband example. When comparing AF488 and AF514, assess how the same green detection window collects each signal.
Confirm settings with measured signals
The peak-position figure cannot compare brightness or calculate compensation. Use measured spectra to inspect filter coverage, then unstained and single-stain samples to assess background and signal. Keep gain or exposure fixed when comparing samples.
References
Chroma · Optical filters and bandwidth
chroma.com
Thermo Fisher · Sources and detection regions
tools.thermofisher.com
AF514 · Labeling & detectionChannel allocation for AF514, AF488 and AF532Neighboring dyes can enter the same detection window. Use single stains to check cross-excitation, spillover and separation.
Channel allocation for AF514, AF488 and AF532
AF514 emission lies between AF488 and AF532, so nearby emission windows can collect neighboring labels. For samples containing GFP or FITC, use single-stain and unstained controls and inspect separation after compensation or unmixing.
Establish separation with controls
Measure each single-stain sample in every relevant channel and use an unstained sample for baseline fluorescence. Set conventional-flow compensation from these measurements or use dye- and treatment-matched references for spectral unmixing. If signals remain poorly resolved, change the dye panel or detection windows.
Comparison of products in similar regions
When comparing dye conjugates of the same target, match target concentration and labeling density and record each source and filter. A brighter reading in one channel may reflect better excitation or collection. Compare specific signal, background, recovery and function alongside raw intensity.
References
Guidelines for the use of flow cytometry and cell sorting in immunological studies (third edition)
Cossarizza A, Chang HD, Radbruch A, et al. · European Journal of Immunology · 51(12):2708–3145 · 2021
Chroma · Optical filters and bandwidth
chroma.com
Labeling guide
Read guide →NHS and maleimide labeling routes
NHS labels primary amines; maleimide labels free thiols. Additives and reduction requirements determine sample preparation.
Labeling guide
Read guide →Azide and DBCO click-reaction pairing
Terminal alkynes pair with azides through CuAAC; DBCO pairs with azides through copper-free SPAAC.
Labeling guide
Read guide →Assessment of AF514-labeled conjugates
Compare labeling density, sample recovery, specific signal and background after purification, rather than total fluorescence alone.
Product-specific documents
Instructions, SDS, product specifications and CoA are catalog-specific. Request documents on the corresponding product page and include the vial lot number for a CoA.
AF514 NHS Ester
QH-10-1005
AF514 Maleimide
QH-10-1005(Maleimide)
AF514 Azide
QH-10-1005(Azide)
AF514-PEG4-DBCO
QH-10-1005(DBCO PEG4)
AF514-PEG12-DBCO
QH-10-1005(DBCO PEG12)
AF514-Annexin V
QH-10-1005(Annexin V)
AF514 WGA
QH-10-1005(WGA)
AF514 CTB
QH-10-1005(CTB)
Spectral and selection support
Include the target, reactive handles, buffer formulation, excitation source and emission window to help identify the product and supporting measurement information.
FAQ
How does the AF514 name relate to excitation and emission?
AF514 NHS has an excitation peak of 517 nm and an emission peak of 542 nm. Check the product name, source wavelength and collection window separately.
Can AF514 and AF488 be used as two independent colors?
Peak separation alone is insufficient. Use measured spectra, instrument channels and single-stain controls; a broad green channel can collect both signals. If separation is unreliable, compare more distant emission regions.
Can AF514 be combined with neighboring dyes?
AF514 emission lies between AF488 and AF532, so nearby emission windows can collect neighboring labels. For samples containing GFP or FITC, use single-stain and unstained controls and inspect separation after compensation or unmixing.
How do NHS, Maleimide, Azide and DBCO match targets?
Use NHS for primary amines, Maleimide for free thiols, Azide for terminal or strained alkynes, and PEG4-DBCO or PEG12-DBCO for azides. Each product page covers pretreatment, buffer and purification requirements.
Can reactive dyes and bioconjugates share storage conditions?
Follow each product page and vial label. Reactive dyes in this catalog are stored dry at −20°C and protected from light; aqueous conjugates have separate requirements. Prepared dye solutions and newly made conjugates cannot simply inherit dry-dye storage conditions.
Can molecular weight, CAS or extinction coefficients be borrowed from a similar dye?
Do not borrow these values. Reactive form, spacer, structure and salt form can affect them; confirm by catalog number. Request missing identity and calculation parameters through the product Documents tab rather than inferring them from a similar name.
How can I obtain instructions, measured spectra and a lot CoA?
Use Documents on the specific product page and describe the application and detection settings. Supply the catalog and vial lot numbers for a CoA. The displayed spectrum is a peak schematic and references concern methods; neither is a lot inspection result.