SKU: 1849081101

Biotin Labeling Efficiency Detection Kit (HABA Method)

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Description

Biotin Labeling Efficiency Detection Kit (HABA Method)Product Specification Synonyms Biotin Labeling Efficiency Detection Kit (HABA Method) Background Principle of the Assay Due to the high binding affinity of biotin for avidin, biotin can displace 4' hydroxyazobenzene 2 carboxylic acid (HABA) from its interaction with avidin, resulting in a proportional decrease in absorbance at 500 nm. The change in absorbance is correlated with the amount of biotin. When a biotin labeled protein solution is added to

Product Specification


Synonyms Biotin Labeling Efficiency Detection Kit (HABA Method)

Background

Principle of the Assay

Due to the high binding affinity of biotin for avidin, biotin can displace 4'-hydroxyazobenzene-2-carboxylic acid (HABA) from its interaction with avidin, resulting in a proportional decrease in absorbance at 500 nm. The change in absorbance is correlated with the amount of biotin.

When a biotin-labeled protein solution is added to the HABA‑avidin mixture, the amount of biotin in the solution can be calculated based on the absorbance change of the HABA‑avidin solution before and after adding the biotin sample, according to the Lambert‑Beer Law.

Applications
Used in conjunction with the biotin rapid conjugation kit to evaluate the biotin labeling efficiency.

Advantages
(1)Reliable — validated, mature and effective assay method
(2)Convenient — the kit provides all reagents required for the assay, including microplates
(3)Flexible — free choice between spectrophotometry or microplate assay

Components

5T(Spectrophotometer Method)
24T(Microplate Reader Method)

Component

Name

Volume

Storage Temperature

Reagent A

HABA Solution

200µL

2°C-8°C

Reagent B

Avidin

2.5mg

2°C-8°C

Reagent C

Phosphate Buffered Saline (PBS)

10mL

2°C-8°C

Component D

Microplate

1 piece

RT


Protocol

Materials Required but Not Provided

Spectrophotometer or microplate reader capable of absorbance detection at 500 nm.

Pipettes covering the range of 20–1000 µL.

 

Reagent Preparation

1. Sample: Prior to analysis, biotin-labeled protein samples must be desalted or dialyzed to remove all unreacted and hydrolyzed biotin conjugating reagent.

2. Preparation of HABA‑Avidin Solution

1) Add 480 µL of Reagent C (Phosphate Buffered Saline, PBS) to the vial containing Reagent B (Avidin) to prepare an avidin solution at 5.2 mg/mL.

2) Calculate the required volume of HABA‑Avidin working solution according to the detection method used (spectrophotometry: 900 µL per test; microplate reader method: 180 µL per well), and prepare accordingly.

3)Prepare HABA‑Avidin working solution::Add 138 µL of Reagent A (HABA solution) and 460 µL of freshly prepared avidin solution to 4002 µL of Reagent C (PBS). Proportional scaling is allowed. Mix thoroughly to obtain the HABA‑Avidin working solution.

4) The absorbance of this solution at 500 nm (A₅₀₀) in a 1‑cm cuvette should be 0.9–1.3.

5) This working solution is stable for two weeks at 4 °C.If precipitation occurs in the HABA solution, filter or centrifuge before use.

 

Measure the absorbance of the labeling system at 500nm using spectrophotometry or microplate enzyme-linked immunosorbent assay as needed.

一、Spectrophotometer Method

1. Add 900 µL of HABA‑Avidin solution into a 1‑cm cuvette, and measure the absorbance at 500 nm, recorded as  A1500.

2. Add 100 µL of biotin‑labeled protein sample into the cuvette containing HABA‑Avidin solution, and mix well.

3. Measure the absorbance of the solution at 500 nm. If the value is ≥ 0.3 and remains constant for at least 15 seconds, record it as A2500.If the value is < 0.3, dilute the biotin‑labeled protein sample and repeat the measurement. The dilution factor must be considered in subsequent calculations.

 

Calculation of Conjugation Efficiency

According to the Beer-Lambert Law: Aλ = ελbC, where:

A :Absorbance of the sample at a specific wavelength (λ). The wavelength used for HABA assay is 500 nm.

ε: Molar extinction coefficient at wavelength (λ). For HABA‑Avidin mixture at 500 nm, ε= 34000 M⁻¹cm⁻¹ at pH 7.0.

b :  Path length (cm). For a 1‑cm cuvette, b = 1 cm.

C : Molar concentration of the sample (mol/L = mmol/mL).

 

Calculations:

1. Calculating the molar concentration of a protein: Protein molar concentration (mmol/mL) = Protein concentration (mg/mL) / Protein molar mass (mg/mmol)

2. Calculate the absorbance change at 500nm of the mixed solution ∆A500: ∆A500 = 0.9 × A1500 - A2500

(Note: 0.9 is the correction factor, which comes from the adjustment of the dilution of the HABA-Avidin mixture by the biotin-labeled protein solution.)

3. Calculate the molar concentration of biotin Biotin(mmol/mL)= ∆A500 / 34000

(注:1 cmcuvette)

4. Calculate the biotin labeling efficiency

Biotin : Protein (molar ratio) = (Biotin molar concentration (mmol/mL) × 10) / (Protein molar concentration (mmol/mL) × Dilution factor)

 

Calculation Example

If the molecular weight of the biotin-labeled antibody is 150,000, the concentration is 3.0 mg/mL, A1500 = 1.0, A2500 = 0.56,then: Molar concentration of the protein: 3/150000 = 2*10-5 mmol/mL

Change in absorbance at 500nm:∆A500= 0.9 ╳ 1.0-0.56=0.34

Molar concentration of biotin: 0.34/34000=1*10-5 mmol/mL

Biotin: protein molar ratio=1*10-5 ╳ 10 /(2*10-5) =5:1

 

二、 Microplate Reader Method

1. Add 180 µL of HABA‑Avidin solution to a microplate well, and measure the absorbance at 500 nm (recorded as A1500)

2. Add 20 µL of biotin‑labeled protein sample to the well containing HABA‑Avidin solution, and mix thoroughly.

3. Measure the absorbance of the solution at 500 nm. Once the value remains constant for at least 15 seconds, record it as  A2500.

 

Calculation of coupling efficiency

According to the Beer-Lambert Law: Aλ = ελbC, where:

A :Absorbance of the sample at a specified wavelength (λ). The wavelength used for HABA assay is 500 nm.

ε:Molar extinction coefficient at wavelength (λ). For the HABA‑avidin mixture at 500 nm, ε = 34000 M⁻¹cm⁻¹ at pH 7.0.

b : Path length (cm). For Component D microplate supplied with the kit, the path height is approximately 0.59 cm at 200 µL.

C : Molar concentration of the sample (mol/L = mmol/mL)。

 

It can be concluded that:

1. Calculating the molar concentration of a protein: Protein molar concentration (mmol/mL) = Protein concentration (mg/mL) / Protein molar mass (mg/mmol)

2. Calculate the change in absorbance of the mixed solution at 500nm∆A500: ∆A500 = A1500-A2500

(Note: Microplates do not require a correction factor because the dilution effect is exactly offset by the increased height of the solution in the wells and the optical path length.)

3. Calculate the molar concentration of biotin:Biotin(mmol/mL)= ∆A500 /(34000 ╳ 0.59)

(Note: The liquid level height/optical path length of the 96-well microplate provided in the kit is为0.59cm)

4. Calculate the biotin labeling efficiency:

Biotin: Protein (molar ratio) = (Biotin molar concentration (mmol/mL) × 10) / (Protein molar concentration (mmol/mL) × Dilution factor)

 

Calculation example

If the molecular weight of the biotin-labeled antibody is 150,000, the concentration is 3.0 mg/mL, A1500 = 0.9, and A2500 = 0.56,then: Molar concentration of the protein: 3/150000 = 2×10⁻⁵ mmol/mL

Change in absorbance at 500nm: ∆A500 = 0.9 - 0.56 = 0.34
Molar concentration of biotin: 0.34 / (34000 × 0.59) = 1.7×10⁻⁵ mmol/mL
Biotin: Protein = 1.7×10⁻⁵ × 10 / (2×10⁻⁵) = 8.5 : 1

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SKU: 1849081101

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