Fluorescein Biotin is a biotin-substituting fluorescent dye that detects and quantifies biotin binding sites by fluorescence or absorbance.Fluorescein Biotin undergoes fluorescence quenching when bound to avidin or streptavidin.

Instructions
1. Solution preparation
Stock solution: Dissolve Fluorescein Biotin in anhydrous DMSO or water to prepare a 1–10 mM stock solution.
Working solution: Dilute to a working concentration of 1–10 µM in assay buffer (e.g., PBS, pH 7.4).
2. Application steps
a. Detection of biotin binding sites
Mix the target protein or molecule (usually avidin or streptavidin conjugated) with the Fluorescein Biotin solution and incubate for 30 minutes to 1 hour at room temperature or 4°C, protected from light.
Wash the sample to remove unbound Fluorescein Biotin (e.g., using PBS containing 0.05% Tween-20).
b. Fluorescence detection
Excitation wavelength: 485–495 nm
Emission wavelength: 515–525 nm
Fluorescence microscopy, flow cytometer, or fluorescence spectrophotometer can be used to detect the signal.
c. Fluorescence quenching study
After mixing the Fluorescein Biotin solution with avidin or streptavidin, observe the change in fluorescence intensity. The degree of quenching is usually related to the binding efficiency and can be used to quantify the density of biotin binding sites.
3. Calibration and control
Set up an unbound Fluorescein Biotin control to distinguish specific binding signals from nonspecific background signals.
Construct a concentration gradient standard curve to calibrate the relationship between the fluorescence signal and the number of binding sites.
4. Precautions
Storage conditions: Fluorescein Biotin should be stored at -20°C in a dark environment and avoid repeated freezing and thawing.
Avoid photobleaching: Avoid strong light exposure during the experiment to avoid weakening the fluorescence signal.
Buffer selection: Use a pH-neutral buffer to ensure the stability of the fluorophore.