Choose dyes across visible and near-infrared wavelengths. Distinguish custom conjugation from direct detection with WGA, CTB or streptavidin before selecting chemistry and detection band.
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Product selection
Select Cy3–Cy7.5 by detection band, reactive dyes by coupling handle, and WGA, CTB or streptavidin by recognition target. Assess labeling density and actual signal after protein coupling.
For support, include: Sample, target handles or recognition target, and detection channel
Coupling and recognition
Cy conjugation: compare input and purified signal
Cy3–Cy7.5 cover different detection bands. Fluorescence after protein coupling also depends on the dye and labeling density; start with a small input series.
The Cy NHS manual gives a protein-labeling reference of about pH 8.5, a 2–5:1 dye:protein molar input and 3–5 hours at room temperature. These are NHS-route starting conditions, adjusted for sample tolerance.
Prepare NHS stock fresh in anhydrous DMSO or DMF as specified. Address Tris, glycine and unintended carrier proteins first. Use the appropriate separate route for maleimide, azide or DBCO.
After removing free dye, assess labeling density with the appropriate extinction and absorbance corrections, alongside target binding, negative background and recovery. More total fluorescence need not mean more effective probe.
WGA already carries a Cy dye and recognizes accessible GlcNAc/sialic-acid-associated glycans.
Dilute according to the aqueous protein-probe formulation. Record live, fixed and permeabilized states and titrate surface staining and uptake separately.
Include unstained and suitable competition controls and record temperature, incubation and localization. Interpret surface-glycan signal separately from intracellular uptake.
Cy–CTB: GM1 binding and trafficking
The CTB probe already carries a Cy label and is handled as a protein probe for GM1-related recognition and trafficking.
Use suitable aqueous dilution and time, temperature and localization controls to distinguish surface binding from uptake. Validate tissue-tracing applications for the model and formulation.
CTB positivity reflects accessible GM1 and probe binding; lipid-raft or membrane-domain conclusions require independent evidence.
Cy–streptavidin: remove free biotin
Cy–streptavidin is a preconjugated biotin-detection probe whose workflow depends on the biotinylated sample.
Remove free biotin and excess biotinylation reagent before detection and check media and formulation additives. Dilute as an aqueous protein probe.
Titrate probe input and include a nonbiotinylated sample. Compare positive separation and background after washing away unbound probe.
These papers and manufacturer methods are selected for the current product use. Their reagents, conditions and parameters belong to the reported systems, not to measured Qihe product performance or universal operating conditions.
Anomalous fluorescence enhancement of Cy3 and cy3.5 versus anomalous fluorescence loss of Cy5 and Cy7 upon covalent linking to IgG and noncovalent binding to avidin
Gruber HJ, Hahn CD, Kada G, et al. · Bioconjugate Chemistry · 11(5):696–704 · 2000
Compares fluorescence changes when Cy3, Cy3.5, Cy5 and Cy7 associate with IgG or avidin, informing joint assessment of labeling density and the purified conjugate’s signal.
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.
A monovalent streptavidin with a single femtomolar biotin binding site
Howarth M, Chinnapen DJF, Gerrow K, et al. · Nature Methods · 3:267–273 · 2006
Uses an engineered monovalent protein to investigate crosslinking by multivalent binding. It informs assessment of probe valency in membrane-protein motion and clustering studies.
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Product manual
Product use, preparation, operating conditions and storage.
Spectra and measurement conditions
Spectra, measurement conditions and detection settings for the exact catalog number.
Certificate of analysis (CoA)
Lot-specific test results. Include the lot number from the vial.
Safety data sheet (SDS)
Handling, storage and emergency response information.
Frequently asked questions
How should I combine Cy color and product-form selection?
Choose the Cy3–Cy7.5 band from actual excitation, emission and detection windows, then decide between conjugation and a WGA, CTB or streptavidin probe. Different forms of the same Cy dye still require catalog-specific parameters.
Can the Cy NHS reference conditions be used for maleimide or click forms?
NHS targets primary amines and maleimide targets free thiols. For azides, distinguish terminal-alkyne CuAAC from strained-alkyne SPAAC; DBCO pairs with azides. Use route-specific buffers, preparation and purification rather than transferring NHS input or timing.
Why can Cy signal or binding fall as labeling density increases?
Fluorescence after protein coupling depends on dye, density and environment; dense labeling can cause quenching or functional loss. Compare purified input-series samples for labeling density, recovery, binding and negative background, separating free-dye effects from overlabeling.
Does Cy-WGA signal directly demonstrate uptake?
WGA recognizes accessible GlcNAc/sialic-acid-associated glycans. Surface binding also fluoresces. Use temperature, incubation, fixation/permeabilization and localization controls to separate surface staining from uptake, and dilute as an aqueous protein probe.
Does Cy-CTB positivity prove the presence of lipid rafts?
CTB signal reflects accessible GM1, probe binding and trafficking. Membrane-domain or lipid-raft conclusions require independent evidence. Separate surface binding from uptake with time, temperature and localization controls; validate tissue tracing for the model and formulation.
What should I check first if Cy-streptavidin signal is weak?
Confirm accessible sample biotin, remove free biotin and excess biotinylation reagent, and check media and formulation additives. Then titrate the aqueous probe against a nonbiotinylated control. Increasing probe input does not replace these checks.
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