Biotinylated antibody detection
Detect bound biotinylated antibodies in suitable immunofluorescence or flow workflows and include antibody-omission controls.
AF532 familyBiotin detection · Fluorescent conjugate
AF532-Streptavidin
Catalog no. QH-15-1005
Fluorescent streptavidin for detecting biotinylated antibodies, nucleic acids and other biotinylated targets.
Excitation530nm
Emission555nm
Binding targetBiotinAccessible biotin
ConjugateAF532–Streptavidin
Spectra and detection conditions ↗Preparation & storage
- Formulation
- Supplied as an aqueous solution
- Storage
- Store at 2–8°C, protect from light. Do not freeze
AF532-Streptavidin
Fluorescent streptavidin for detecting biotinylated antibodies, nucleic acids and other biotinylated targets.
Streptavidin recognizes biotin through noncovalent affinity binding. Residual free biotin competes for binding sites, and endogenous biotin can contribute background.
AF532–Streptavidin
Biotin
Biotin affinity binding
Dashed line: noncovalent binding.
Applications and samples
Biotinylated nucleic acids and probes
Check biotin accessibility, post-hybridization washing and off-target background.
Formulation and use
Supplied at 2 mg/mL in PBS (pH approximately 7.4), with 2 mg/mL BSA and 0.05% sodium azide. The suggested working concentration is 1–2 μg/mL, with sample-specific titration. Prepare working solutions fresh.
AF532-Streptavidin · Specifications
- Product name
- AF532-Streptavidin
- Catalog no.
- QH-15-1005
- Dye family
- AF532
- Conjugated ligand
- Streptavidin
- Binding target
- Accessible biotin
- Pack sizes
- 1 mg / 5 mg / 10 mg / 25 mg / 100 mg
- Formulation
- Supplied as an aqueous solution
- Storage
- Store at 2–8°C, protect from light. Do not freeze
- Excitation peak (catalog reference)
- 530 nm
- Emission peak (catalog reference)
- 555 nm
- Ligand name
- Streptavidin
- Binding mode
- Noncovalent biotin–streptavidin affinity binding
- Stock concentration
- 2 mg/mL
- Volume of the 1 mg pack
- 0.5 mL
- Buffer
- PBS, pH approximately 7.4
- Stabilizer
- 2 mg/mL BSA (IgG- and protease-free)
- Preservative
- 0.05% sodium azide
- Storage stability
- At least 2 years at 2–8°C, protected from light
- Shipping
- Ship with cold packs, protected from light
- Suggested working concentration
- 1–2 μg/mL; titrate for the sample
- Working solution
- Prepare fresh; avoid prolonged storage after dilution
Concentration, volume and optical measurements
A 2 mg/mL stock diluted to 1–2 μg/mL corresponds to approximately 1:2000–1:1000. Determine the working amount from sample signal and background. Contains sodium azide; for in vitro research only.
AF532-Streptavidin · Protocol
The following outlines assay preparation. Confirm amounts, incubation time and temperature using this product’s instructions.
Before staining
Prepared 0 / 3
Review upstream labeling
Confirm target identity, biotinylation method and function; for in-house biotinylation, review removal of unbound reagent.
Address background sources
Assess endogenous biotin and nonspecific binding and use a blocking approach compatible with the detection workflow.
Set the working amount
The stock concentration is 2 mg/mL; use 1–2 μg/mL as the suggested starting range for titration. Keep the upstream biotinylated probe amount fixed and compare signal and background.
Bind and wash
Incubate protected from light and remove unbound conjugate using the appropriate method; match temperature, time and washing across groups.
Acquire and analyze
Check target-associated signal against negative controls and use single-stain compensation or unmixing controls for multicolor detection.
Assay troubleshooting
Weak signal on a biotinylated target
Check free-biotin competition, inaccessible sites, upstream probe binding and channel settings.
Signal without the upstream antibody
Assess endogenous biotin, nonspecific conjugate adsorption, amount and washing.
Request product documentation
Choose a document and enter your phone and email. Include the product lot number for a CoA.
AF532-StreptavidinQH-15-1005
Product manual
Working solutions, assay conditions, sample requirements and storage
Certificate of analysis (CoA)
Provide the vial lot number to request the corresponding test items and results
Safety data sheet (SDS)
Formulation-specific handling, protection and disposal information
Supplementary documents
Streptavidin formulation and use
Formulation, storage and working-solution preparation
Fluorescent streptavidin selection catalog
Catalog numbers, peaks and pack sizes for 13 AF conjugates
Formulation and labeling information
Concentration, buffer, preservatives, labeling density and available testing records
Application and detection information
Sample preparation, controls, assay guidance and troubleshooting
Batch quality
Catalog specifications identify the product. Purity, component analysis, test methods and acceptance criteria are supplied in the lot-matched CoA. Include the vial lot number when requesting the report above.
Information for technical support
Include sample type, live or fixed state, buffer, instrument and channels, existing controls and the question to resolve.
Technical articles
Labeling guide
Read guide →Effects of free biotin on detection
Free biotin occupies streptavidin-binding sites and can reduce detection of biotinylated targets.
Labeling guide
Read guide →Analysis of endogenous biotin and nonspecific background
Use unstained, streptavidin-only and non-biotinylated-probe controls to distinguish autofluorescence, endogenous biotin and adsorption.
Labeling guide
Read guide →Streptavidin fluorescence detection and multivalent binding
Multivalent streptavidin can crosslink biotinylated targets, affecting live-cell motion and clustering measurements.
Spectra & detection
Read article →AF532 spectra and detection channels
Read the bandpass wavelength range and use single-stain controls to check spillover.
References
References on reaction chemistry, ligand recognition and assay interpretation. Reagents and conditions belong to each study; these papers do not validate Qihe AF product performance or establish equivalence to Alexa Fluor products.
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.
DOI: 10.1038/nmeth861
FAQ
How should a 2 mg/mL stock be diluted?
A 2 mg/mL stock diluted to 1–2 μg/mL corresponds to approximately 1:2000–1:1000. Calculate from the actual vial concentration and titrate on the sample. Prepare diluted working solutions fresh.
Does the target need biotin?
Detection requires accessible biotin, supplied by a biotinylated antibody, nucleic acid or other probe.
Is this monovalent streptavidin?
The product documentation does not specify a monovalent form. Contact technical support if valency is critical.
How should I store it? Can I freeze it?
Store at 2–8°C, protect from light. Do not freeze. Consult the instructions for the storage duration of diluted working solutions.
How can I confirm concentration, labeling density and lot quality?
The stock concentration is 2 mg/mL; buffer and additives are listed in Specifications. Use the vial lot number to request the corresponding CoA for labeling density and test results.
Documents →