How much biotin is coupled to my protein? The HABA/Avidin Assay

Biotin, a 244 Dalton molecule, exhibits an extraordinary binding affinity for avidin and streptavidin (K_a=10¹⁵ M^-1). This high binding affinity means that proteins, particularly antibodies, are routinely labeled with biotin.  The labeled proteins can now be rapidly purified with avidin and streptavidin resins or detected with streptavidin and avidin coupled to enzyme reporters, such as HRP (horseradish peroxidase.

The selection selection of a biotinylation reagent and the coupling chemistries are covered in these two blogs:

• Biotin Labeling: Key Points to Selecting Your Biotin Agent
• The Secrets of Coupling with Biotin!

This blog's focus is how do you determine the actual amount of biotin coupled to your protein or how do you determine if biotinylation was successful. 

The most common method is to employ HABA (2-(4-Hydroxyphenylazo)benzoic acid/ 2-(4′-Hydroxybenzeneazo)benzoic acid/ 4′-Hydroxyazobenzene-2-carboxylic acid) to rapidly estimate the mole-to-mole ratio of biotin to antibody or protein.  The method of biotin incorporation estimation is based on the binding of avidin with HABA dye, which produces a color that can be read at 500nm. The HABA-avidin complex can be displaced with free biotin or biotin conjugated with other molecules (proteins). Measuring the change in optical density of HABA-avidin complex with biotinylated proteins allows for accurate estimation of the molar ration of biotin conjugated to the protein/ antibody.

The following procedure is adapted from our HOOK™ BiotinQuant™ protocol.

Protocol

1. First remove all the free or unconjugated biotin from the labeled protein to ensure accurate results.  This can be removed by gel filtration or desalting columns or dialysis
2. Prepare a solution of HABA/Avidin:
   1. Add 12.1mg HABA to 4.95ml pure water and then add 50μl 1N NaOH. Vortex to dissolve and filter if required.
   2. Add 5mg avidin and 300μl above HABA solution to 9.7ml PBS
3. Pipette 900μl HABA/Avidin solution into a 1ml cuvette and measure the absorbance at 500nm and record the value
4. Add 100μl biotinylated protein sample to the above cuvette and mix well.
5. Measure the absorbance at 500nm and once value remains steady for 15 seconds record the result.
   NOTE: For microplates use 180μl HABA/Avidin and 20μl sample.
   

Calculation

The calculation is based on Beer Lambert (Beer’s) Law: A_λ = ε_λbC, where

• A is the absorbance at a particular wavelength (λ)
• ε is the extinction coefficient at the wavelength (λ). For HABA/Avidin samples at 500nm, pH7.0 this is 34,000M^-1cm^-1..
• b is the path length in centimeters. Cuvettes (10x10mm) have a pathlength of 1cm. The pathlength for microplates, using the indicated volumes, is normally 0.5cm.
• C is the molarity concentration of the sample (= mol/L = mmol/ml)

For calculating the number of moles of biotin per mole of protein or sample the following values are required:

• Concentration of protein/sample used (mg/ml)
• Molecular weight of protein, expressed as grams per mole (e.g. IgG = 150,000)
• A₅₀₀ HABA/Avidin reading
• A₅₀₀ HABA/Avidin/Biotin Sample
• Dilution factor (DF), if sample was diluted before adding to HABA/avidin solution.

1. Calculate mmol biotinylated protein/ml

protein concentration (mg/ml) ÷ MW of protein (mg/mmol)

2. Calculate change in absorbance at 500nm

For Cuvette: (0.9 x A₅₀₀ HABA/Avidin) –( A₅₀₀ HABA/Avidin/Biotin Sample) = ΔA₅₀₀

For Microplate: (A₅₀₀ HABA/Avidin) –( A₅₀₀ HABA/Avidin/Biotin Sample) = ΔA₅₀₀ 

NOTE: 0.9 is the correction factor for the dilution of the HABA/Avidin with the sample in the cuvettes. This is not necessary for microplates as the dilution is offset by the increase in volume and therefore the light path (b).

3. Calculate concentration of biotin in reaction (mmol/ml)

ΔA₅₀₀ (Calculation # 2) ÷ (34,000 x b)

NOTE: b = lightpath, which is 1cm for cuvettes and 0.5cm for microplates.

4. Calculate mmol of biotin per mmol of protein

(mmol biotin in reaction (Calculation # 3) x 10 x DF) ÷ mmol protein in original sample (Calculation # 1)

NOTE: DF is the dilution factor. 10 is for the 10 fold dilution of the biotinylated protein sample in the reaction mixture.