Effects of free biotin on detection
Free biotin occupies streptavidin-binding sites and can reduce detection of biotinylated targets.
Applies to: For prepared streptavidin probes; check competition from free biotin.
Streptavidin probes for biotin detection
Choose a ready-labeled probe for biotinylated samples. Confirm biotin accessibility and removal of free biotin, then compare small-dye, fluorescent-protein and tandem formats.
24 products
Product selection
For affinity detection of biotinylated samples. Confirm accessible biotin and remove free biotin before choosing the fluorescent format and detection band.
24 matching products
| Product / Cat. No. | Excitation / nm | Emission / nm | Pack sizes | Product details |
|---|---|---|---|---|
| AF350-StreptavidinQH-15-1001 | 346 | 445 | 1 mg / 5 mg / 10 mg / 25 mg / 100 mg | |
| AF405-StreptavidinQH-15-1002 | 400 | 424 | 1 mg / 5 mg / 10 mg / 25 mg / 100 mg | |
| AF430-StreptavidinQH-15-1003 | 430 | 545 | 1 mg / 5 mg / 10 mg / 25 mg / 100 mg | |
| RPE-Cy5-StreptavidinQH-15-1019 | 488 | 670 | 1 mg / 5 mg / 10 mg / 25 mg / 100 mg | |
| Percp-Cy5.5-StreptavidinQH-15-1021 | 489 | 679 | 1 mg / 5 mg / 10 mg / 25 mg / 100 mg | |
| AF488-StreptavidinQH-15-1004 | 494 | 517 | 1 mg / 5 mg / 10 mg / 25 mg / 100 mg | |
| AF532-StreptavidinQH-15-1005 | 530 | 555 | 1 mg / 5 mg / 10 mg / 25 mg / 100 mg | |
| AF546-StreptavidinQH-15-1006 | 554 | 570 | 1 mg / 5 mg / 10 mg / 25 mg / 100 mg | |
| Cy3-StreptavidinQH-15-1014 | 554 | 564 | 1 mg / 5 mg / 10 mg / 25 mg / 100 mg | |
| AF555-StreptavidinQH-15-1007 | 555 | 572 | 1 mg / 5 mg / 10 mg / 25 mg / 100 mg | |
| PE-Cy5.5-StreptavidinQH-15-1018 | 565 | 700 | 1 mg / 5 mg / 10 mg / 25 mg / 100 mg | |
| R-PE-StreptavidinQH-15-1023 | 565 | 575 | 1 mg / 5 mg / 10 mg / 25 mg / 100 mg | |
| RPE-Cy7-StreptavidinQH-15-1020 | 565 | 780 | 1 mg / 5 mg / 10 mg / 25 mg / 100 mg | |
| AF568-StreptavidinQH-15-1008 | 578 | 602 | 1 mg / 5 mg / 10 mg / 25 mg / 100 mg | |
| AF594-StreptavidinQH-15-1009 | 590 | 617 | 1 mg / 5 mg / 10 mg / 25 mg / 100 mg | |
| Cy5-StreptavidinQH-15-1015 | 640 | 664 | 1 mg / 5 mg / 10 mg / 25 mg / 100 mg | |
| APC-StreptavidinQH-15-1024 | 650 | 665 | 1 mg / 5 mg / 10 mg / 25 mg / 100 mg | |
| AF647-StreptavidinQH-15-1010 | 651 | 672 | 1 mg / 5 mg / 10 mg / 25 mg / 100 mg | |
| APC-AF700-StreptavidinQH-15-1022 | 651 | 780 | 1 mg / 5 mg / 10 mg / 25 mg / 100 mg | |
| AF660-StreptavidinQH-15-1011 | 668 | 698 | 1 mg / 5 mg / 10 mg / 25 mg / 100 mg | |
| Cy5.5-StreptavidinQH-15-1016 | 680 | 698 | 1 mg / 5 mg / 10 mg / 25 mg / 100 mg | |
| AF680-StreptavidinQH-15-1012 | 684 | 707 | 1 mg / 5 mg / 10 mg / 25 mg / 100 mg | |
| AF700-StreptavidinQH-15-1013 | 702 | 723 | 1 mg / 5 mg / 10 mg / 25 mg / 100 mg | |
| Cy7-StreptavidinQH-15-1017 | 740 | 770 | 1 mg / 5 mg / 10 mg / 25 mg / 100 mg |
For support, include: Biotinylated sample, free-biotin removal, detection channel and background issue
Affinity detection and background
Remove free biotin before adding the probe
Streptavidin binds accessible biotin. Excess biotinylation reagent, free biotin and endogenous biotin can affect detection.
- Purify the biotinylated sample first and check media, blocking solution and formulation additives for biotin that could occupy probe-binding sites.
- Allow the biotinylated primary antibody or ligand to bind, wash away unbound material, then add this probe and wash as required. Validate background separately for other assembly orders.
- Use probe titration and a nonbiotinylated control to distinguish signal from background. In receptor or particle systems, assess aggregation or crosslinking associated with multivalent binding.
Handle small-dye, protein and tandem formats appropriately
All three formats are preconjugated detection probes; working concentration and storage depend on the actual formulation.
- AF/Cy small-dye conjugates still contain streptavidin protein. Dilute as aqueous protein probes rather than applying DMSO reconstitution intended for free dyes.
- Protect the fluorescent-protein state of R-PE, APC and tandem formats. Tandem single-stain references must also match lot and fixation/permeabilization.
Method resources
Read article
Analysis of endogenous biotin and nonspecific background
Use unstained, streptavidin-only and non-biotinylated-probe controls to distinguish autofluorescence, endogenous biotin and adsorption.
Applies to: For biotin-affinity detection; distinguish endogenous biotin from nonspecific adsorption.
Streptavidin fluorescence detection and multivalent binding
Multivalent streptavidin can crosslink biotinylated targets, affecting live-cell motion and clustering measurements.
Applies to: For assessing probe-induced crosslinking; the cited engineered monovalent protein does not establish that current products are monovalent.
References and method background
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.
Thermo Fisher · Biotin binding and interference
Thermo Fisher Scientific
Sample preparation, free biotin and background control in biotin-affinity detection.
Thermo Fisher · Fluorescent streptavidin methods
Thermo Fisher Scientific
Sample preparation, free biotin and background control in biotin-affinity detection.
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
Tandem-dye donor leakage, processing stability and controls
R&D Systems · Tandem Dye-Conjugated Antibodies FAQs
The source concerns conjugated antibodies and informs energy transfer, degradation and lot-matched controls. Qihe’s tandem series contains labeling reagents; validate the final conjugate with lot- and process-matched single stains. The source is not a raw-reagent conjugation recipe.
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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
Why can a biotinylated sample still give little signal?
Check biotin accessibility and remove free biotin and excess biotinylation reagent. Free biotin in media, blocking solution or formulation additives can occupy streptavidin sites before detection. Then check the channel and probe amount.
What is the usual order for a biotinylated primary antibody and fluorescent streptavidin?
Bind the biotinylated primary antibody or ligand to the sample, wash away unbound material, then add fluorescent streptavidin and wash. Validate background and multivalent aggregation separately for premixing or other assembly orders.
Can AF/Cy-streptavidin be prepared in DMSO like a free dye?
These products contain streptavidin protein and are preconjugated detection probes. Use the formulation’s aqueous dilution instructions rather than an organic-stock procedure for a free reactive AF/Cy dye.
What needs extra attention for PE, APC and tandem streptavidin?
In addition to biotin accessibility, protect the fluorescent-protein state and follow the actual formulation’s storage requirements. Use lot- and processing-matched single stains for tandem formats; working amounts and references are not interchangeable across carriers.
How should high background or cell/particle aggregation be assessed?
Check background with a nonbiotinylated sample and assess endogenous biotin, washing and probe input. Multivalent streptavidin binding can bridge biotin sites; in receptor or particle systems, compare aggregation before and after probe addition.