Which groups are on your target?
Consider NHS for primary amines and Maleimide for free thiols. For click chemistry, confirm the reaction partners first.
AF350 fluorescent dyes
Blue emission, with 5 reactive dyes and 4 bioconjugates. UV excitation and blue collection; find labeling methods and documents for the target handle.
346nm
445nm
9
Optical values refer to AF350 NHS.
AF350 NHS spectral referenceBlue
AF350 product selection
Find AF350 dyes and conjugates by reactive group or recognition ligand.
Which recognition ligand do you need?
Compare Annexin V, WGA, CTB, Streptavidin; check the sample and conditions.
| Product / catalog no. | Reactive form / ligand | Targets and applications | Documents |
|---|---|---|---|
| AF350 NHS EsterQH-10-1001 | NHS | Antibodies, proteins and other molecules with accessible primary amines | View product → |
| AF350 MaleimideQH-10-1001(Maleimide) | Maleimide | Targets with accessible free thiols | View product → |
| AF350 AzideQH-10-1001(Azide) | Azide | Targets with a compatible alkyne; confirm the click reaction system | View product → |
| AF350-PEG4-DBCOQH-10-1001(DBCO PEG4) | PEG4-DBCO | Azide coupling with a PEG4 spacer | View product → |
| AF350-PEG12-DBCOQH-10-1001(DBCO PEG12) | PEG12-DBCO | Azide coupling; evaluate a PEG12 spacer | View product → |
| AF350-Annexin VQH-10-1001(Annexin V) | Annexin V | Recognizes exposed PS; binding requires Ca²⁺ | View product → |
| AF350 WGAQH-10-1001(WGA) | WGA | Recognizes glycans containing GlcNAc or sialic acid | View product → |
| AF350 CTBQH-10-1001(CTB) | CTB | Recognizes GM1; consider binding and uptake conditions | View product → |
| AF350-StreptavidinQH-15-1001 | Streptavidin | Detect existing biotin labels using Streptavidin | View product → |
Showing 9 / 9 AF350 products
Spectra and detection conditions
AF350 NHS optical properties
- Reference product
- AF350 NHS · QH-10-1001
- Excitation peak
- 346 nm
- Emission peak
- 445 nm
- Molar extinction coefficient
- 19,000 M⁻¹ cm⁻¹
Values refer to this product’s specifications. Consult measurement records for solvent, pH and conjugation state.
Full product specifications →UV excitation and blue collection
Check for a UV excitation path around 350 nm and transmission through the sample vessel, objective and optics in this region. Blue emission alone does not establish compatibility with an instrument that only has a 405 nm laser.
A 450/50 bandpass nominally spans 425–475 nm and includes the 445 nm emission peak. Collected signal depends on the full emission spectrum, filter transmission and detector response; this is a passband example. A DAPI channel label does not specify the excitation bandwidth; inspect both UV excitation and blue collection.
Read the detection guide →Multicolor combinations and controls
AF350 (445 nm) and AF405 (424 nm) may share short-wavelength collection regions. Different excitation sources do not remove the need to assess detection spillover. With DAPI or another nuclear stain, record single-stain signals under each excitation path before choosing the panel.
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 |
|---|---|
| AF350-Annexin V | Phosphatidylserine; binding is Ca²⁺ dependent. |
| AF350 WGA | GlcNAc- or sialic-acid-containing glycans; consider sample preparation and intracellular signal. |
| AF350 CTB | GM1 ganglioside; confirm the application and treatment conditions. |
| AF350-Streptavidin | Biotinylated targets; account for competing free biotin. |
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
AF350 · Labeling & detectionAF350 spectra and detection channelsAF350 NHS has 346/445 nm peaks. Check the excitation source, emission window and detector response separately.
Product peaks and excitation source
AF350 NHS has an excitation peak of 346 nm and an emission peak of 445 nm. Check the product name, source wavelength and collection window separately. Check for a UV excitation path around 350 nm and transmission through the sample vessel, objective and optics in this region. Blue emission alone does not establish compatibility with an instrument that only has a 405 nm laser.
Emission window and optical path
A 450/50 bandpass nominally spans 425–475 nm and includes the 445 nm emission peak. Collected signal depends on the full emission spectrum, filter transmission and detector response; this is a passband example. A DAPI channel label does not specify the excitation bandwidth; inspect both UV excitation and blue collection.
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
AF350 · Labeling & detectionAF350 and AF405 spectral comparisonNeighboring dyes can enter the same detection window. Use single stains to check cross-excitation, spillover and separation.
AF350 and AF405 spectral comparison
AF350 (445 nm) and AF405 (424 nm) may share short-wavelength collection regions. Different excitation sources do not remove the need to assess detection spillover. With DAPI or another nuclear stain, record single-stain signals under each excitation path before choosing the panel.
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 AF350-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.
AF350 NHS Ester
QH-10-1001
AF350 Maleimide
QH-10-1001(Maleimide)
AF350 Azide
QH-10-1001(Azide)
AF350-PEG4-DBCO
QH-10-1001(DBCO PEG4)
AF350-PEG12-DBCO
QH-10-1001(DBCO PEG12)
AF350-Annexin V
QH-10-1001(Annexin V)
AF350 WGA
QH-10-1001(WGA)
AF350 CTB
QH-10-1001(CTB)
AF350-Streptavidin
QH-15-1001
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 AF350 name relate to excitation and emission?
AF350 NHS has an excitation peak of 346 nm and an emission peak of 445 nm. Check the product name, source wavelength and collection window separately.
Can AF350 be assigned directly to a 405 nm channel?
First establish AF350 excitation at that source wavelength. Its name and blue emission do not replace excitation requirements. If UV optics are unavailable, compare AF405 and reevaluate the conjugate.
Can AF350 be combined with neighboring dyes?
AF350 (445 nm) and AF405 (424 nm) may share short-wavelength collection regions. Different excitation sources do not remove the need to assess detection spillover. With DAPI or another nuclear stain, record single-stain signals under each excitation path before choosing the panel.
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.