Biotin, also known as vitamin H, is a small molecule that is present in tiny amounts in all living cells and is critical for a number of biological processes. Due to the low molecular weight of biotin, biotin usually does not cause any significant changes in protein conformation and biological activity. Therefore, biotin is widely applied for biotin-labeling of antibodies and other proteins of interest.
The Aladdin Biotin Quantitation Kit (HABA, Colorimetric Method) permits rapid quantification of biotin-labeling level for antibodies, proteins and other biomolecules, as well as biotin concentrations within test samples. HABA, chemically designated as 4'-hydroxyazobenzene-2-carboxylic acid, is a small-molecular-weight dye capable of weak intermolecular interaction with streptavidin (SA). Assay Principle: HABA associates with streptavidin (SA) to yield a complex exhibiting an absorption maximum at 500 nm. Because of its higher affinity for avidin, biotin displaces the HABA and the absorbance at 500nm decreases proportionately. Accordingly, the presence of free biotin or biotin-conjugated proteins in solution produces a reduction in absorbance at 500 nm. The kit provides HABA/Streptavidin Premix, biotin standard, positive control, and reaction buffer. Both the absorbance difference-value method and biotin standard curve method can be used for biotin quantification and calculation of the biotin-to-protein molar ratio. When using the biotin standard curve method, the kit detects biotin solutions over the range of 2-40 μM (400 pmol-8000 pmol). This kit is available in two sizes: 100-assay and 500-assay.
Contents and storage
| Component No. | Component Name | Appearance | 100T | 500T | Storage | Quantity Per Test |
| B1524815A | HABA/Streptavidin Premix | liquid | 2 mL | 10 mL | -20℃. Store in the dark. | 20 μL for 1 test |
| B1524815B | Reaction Buffer | liquid | 20 mL | 100 mL | -20℃. | 160 μL for 1 test |
| B1524815C | Biotin Standard (500μM) | liquid | 1 mL | 5 mL | -20℃. Store in the dark. | Use as needed
|
| B1524815D | BSA-Biotin (Positive Control)
| liquid | 0.5 mL | 1.5 mL | -20℃. Store in the dark. | Use as needed
|
Required materials not supplied
1. 96-well clear flat-bottom microplate.
2. Multi-well spectrophotometer (ELISA reader).
3. Microcentrifuge.
Important
1. The HABA/Streptavidin Premix is light-sensitive. Store protected from light and use promptly after reconstitution.
2. Aliquot the reagent for storage; avoid repeated freeze-thaw cycles.
3. Avoid potassium-containing buffers; potassium ions induce precipitation within the reaction system. Dissolve samples in potassium-free PBS (sodium phosphate) or TBS.
4. Do not include reducing agents such as DTT or β-mercaptoethanol in the reaction system; these agents disrupt streptavidin structure and cause aberrant absorbance readings.
5. Should precipitate appear after thawing Reaction Buffer stored at -20 °C, incubate at 37 °C in a water bath until completely dissolved.
6. BSA-Biotin serves only as a biotin-labeling positive control. No value for the biotin-to-protein ratio is supplied.
7. Before formal experimentation, the reagents must be returned to room temperature and centrifuged before use. Both lower temperatures and reagent residue on the centrifuge tube walls may impair conjugation efficiency.
8. During operation, always wear a lab coat, disposable gloves, and protective equipment.
9. All products are for research use only.
Instruction for use
1.Absorbance difference method
1.1 Add 20 μL of HABA/SA premix followed by 160 μL of reaction buffer to a 96-well clear flat-bottom microplate. Mix gently and avoid bubble formation.
1.2 Read the absorbance at 500 nm using a microplate reader; record this value as A₅₀₀HABA/SA(A₅₀₀H\S).
1.3 Add 20 µL of target biotinylated protein sample, BSA-Biotin(Positive Control), Reaction Buffer(Negative control). Mix gently and avoid bubble formation.
1.4 Read the absorbance at 500 nm using a microplate reader; record this value as A₅₀₀HABA/SA/Biotin(A₅₀₀H\S\B).
Note: If A₅₀₀H\S\B)<0.15, dilute the biotinylated antibody or protein with reaction buffer and retest. If A₅₀₀H\S\B)>0.43, concentrate the biotinylated antibody or protein and retest.
2. Direct Quantitation
Recommended: Aladdin provides a downloadable Biotin Quantitation Calculation Sheet. Simply input all measured values to automatically obtain the biotin-to-antibody or biotin-to-protein molar ratio.
2.1 Calculation for the change in absorbance at 500 nm
∆A₅₀₀=A₅₀₀H\S-A₅₀₀H\S\B
2.2 Calculation for the molar concentration of biotin in the reaction system
Based on the Beer-Lambert Law, the biotin concentration in the diluted system is calculated from the absorbance change.
Biotin(mM)=∆A₅₀₀/(34500× L (cm) )
Where:
The extinction coefficient of the HABA-SA mixture at 500 nm is ε ( M⁻¹ cm⁻¹ ) =34500 M⁻¹ cm⁻¹.
L (cm) is the cell path length expressed in centimeters (cm). For a 96-well microplate with 200 μL solution, the path length is typically 0.5 cm.
2.3 Calculation for the molar concentration of the original protein sample
Protein(mM)=protein concentration (mg/mL)/MW of protein (Da)
2.4 Calculation for the mmol of biotin per mmol of protein
Calculation for correcting the sample dilution factor to obtain the average number of biotin molecules conjugated per protein molecule:
Biotin(mM)/Protein(mM)=Biotin(mM)×10×Dilution factor/Protein(mM)
Where:
10 refers to the dilution factor of the system. 20 μL sample plus 180 μL reaction mixture results in a 10-fold dilution of the sample.
The dilution factor is the extra dilution applied to biotinylated antibody/protein when high initial concentration leads to A₅₀₀H\S\B < 0.15; use 1 if no dilution is done.
3. Standard-Curve Calibration
To avoid potential errors in the extinction coefficient and path length, the biotin standard curve method may be used.
3.1 Standard Curve Preparation
Add directly to the 96-well microplate using the volumes in the table below:
Tube | Volume of Biotin Standard (500μM) (μL) | Reaction Buffer (μL) | Final Biotin Concentration (µM) | Final Biotin Amount (nmol)¹ |
1² | 0 | 20 | 0 | 0 |
2 | 1 | 19 | 2.5 | 0.5 |
3 | 2 | 18 | 5 | 1 |
4 | 4 | 16 | 10 | 2 |
5 | 6 | 14 | 15 | 3 |
6 | 8 | 12 | 20 | 4 |
7 | 10 | 10 | 25 | 5 |
8 | 12 | 8 | 30 | 6 |
9 | 14 | 6 | 35 | 7 |
10 | 16 | 4 | 40 | 8 |
Note¹: The reaction system volume is 200 μL. The calculated total biotin content (nmol) represents the biotin amount contained in the 20 μL sample/standard added per well.
Note²: Blank control.
3.2 Sample addition: Add 20 μL of sample, BSA-Biotin (positive control), and standards from tubes 1 to 10 of each gradient to respective wells of the microplate.
3.3 Measurement
3.3.1 Add the corresponding solutions to each well of the microplate according to the table below.
Reagent Name | Sample Tube | Positive Control | Standard Tube |
Reaction Buffer | 160 μL | 160 μL | 160 μL |
HABA/Streptavidin Premix | 20 μL | 20 μL | 20 μL |
Biotin Standard (500μM) | 0 μL | 0 μL | 20 μL³ |
BSA-Biotin (Positive Control) | 0 μL | 20 μL | 0 μL |
Sample | 20 μL | 0 μL | 0 μL |
Note³: Prepare each gradient standard from Tube 1 to Tube 10 following the table in Section 3.1.
3.3.2 Mix gently to avoid bubble formation, then read the absorbance at 500 nm using a microplate reader.
Note: If A₅₀₀H\S\B)<0.15, dilute the biotinylated antibody or protein with reaction buffer and retest. If A₅₀₀H\S\B)>0.43, concentrate the biotinylated antibody or protein and retest.
3.4 Data Analysis
3.4.1 Plot the Biotin standard curve with the amount of Biotin (nmol) on the X-axis and ∆ A₅₀₀ standard = A₅₀₀ blank- A₅₀₀ standard on the Y-axis (linear relationship, see Figure 1), and fit to obtain the equation. Use the equation to calculate the total molar amount of biotin (nmol) in samples.
Figure 1 Schematic diagram of the Biotin standard curve for Aladdin Biotin Quantitation Kit (HABA, Colorimetric Method) (B1524815). Actual test data may vary depending on experimental conditions, detection instruments and other factors; the data shown in the figure are for reference only.
3.5 Calculation for the mmol of biotin per mmol of protein
Biotin-to-protein molar ratio=n(Biotin)/n(Protein)
n(Protein)=(c×20)/(M×10⁶)
Where:
c is the concentration of biotinylated protein (mg/mL).
20 is the sample loading volume (μL).
M is the molecular weight of the protein (g/mol)
Note: n(Biotin) refers to total biotin of the sample in the reaction well, and n(Protein) refers to total protein of the sample in the reaction well. The two values must be consistent.
4. The calculated result represents the biotin-to-protein molar ratio, i.e., the average number of biotin molecules conjugated to each protein molecule. The optimal labeling range for IgG antibodies is 2.0-6.0. Over-labeling will reduce antibody activity, whereas under-labeling leads to weak signals.
