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Amplite® Rapid Colorimetric Biotin Quantitation Kit *Optimized to Use with Nanodrop*

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Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
StorageFreeze (< -15 °C); Minimize light exposure
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OverviewpdfSDSpdfProtocol


Amplite™ Colorimetric Biotin Quantitation Kit is designed to quantify the number of biotins attached to a protein or other macromolecule with a Nanodrop Spectrometer. The kit uses HABA (4’-hydroxyazobenzene-2-carboxylic acid) reagent that demonstrates a dramatic spectral change when bound to avidin. Biotin easily displaces HABA from the HABA/avidin complex, resulting in a decrease of absorption at 500 nm. By measuring the absorbance change before and after the addition of the biotin-containing sample at 500 nm, the amount of biotin in a sample can be quantified spectrophotometrically. The kit is best used to determine biotin concentration in the range from 2 to 16 µM. It is optimized to use with Nanodrop spectrometer. Biotin is a relatively small molecule that is routinely conjugated to antibodies and proteins with minimal interference of their biological activity. The avidin/streptavidin-biotin interaction is the strongest known binding pair between a protein and its ligand. The biotin-avidin interaction has been extensively explored for a variety of biological applications.

Components


Example protocol


PREPARATION OF STOCK SOLUTIONS

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles

100X Avidin Stock Solution
  1. To prepare a 100X Avidin stock solution, add 40 µL of ddH2O into the vial of Avidin (Component A), and mix thoroughly.

    Note: Any unused 100X Avidin stock solution should be divided into single-use aliquots and stored at -20°C.

PREPARATION OF WORKING SOLUTION

HABA/Avidin Working Solution
  1. To prepare a HABA/avidin working solution, add 2 µL of the 100X Avidin stock solution into 200 µL of the HABA assay buffer (Component B), and mix thoroughly.

    Note: Any unused HABA/avidin working solution can be store for up to one week at 4°C.

SAMPLE EXPERIMENTAL PROTOCOL

Quantitate Amount Biotin with a Nanodrop
  1. Prepare the following controls and samples as outlined in the table below:

    Positive ControlNegative ControlTest Sample*
    d-Biotin (Component C): 2 µLddH2O: 2 µL2 µL
    HABA/Avidin working solution: 18 µLHABA/Avidin working solution: 18 µLHABA/Avidin working solution: 18 µL

    Note: To ensure the concentration of biotin falls within the assay's linear range (2-16 μM of biotin final concentration), it is necessary to test the biotin-containing samples at several dilutions.

    Note: To ensure assay accuracy, avoid using buffers containing potassium, as it causes precipitation.

    Note: Free biotin must be separated from the biotinylated protein by gel filtration or dialysis.

  2. Incubate the reaction mixture at room temperature for 5 minutes, protected from light. Avoid creating bubbles during pipetting.

  3. Monitor absorbance at 500 nm using a Nanodrop Spectrometer, with a sample measurement of 2~2.5 µL. Utilize the protein label function for readings.

DATA ANALYSIS - CALCULATIONS
1. Calculate the change of absorbance at 500 nm:

ΔA500 = A500 of negative control - A500 of Biotin sample or positive control

2. Calculate the biotin concentration (M):

M = [ΔA500 ÷ εHABA/Biotin] x DF

Where:

  • εHABA/Biotin = 34,500 M-1cm-1
  • DF = Dilution Factor
3. Calculate protein concentration (P)

P = protein concentration (mg/mL) ÷ molecular weight of protein

4. Calculate molar ratio of biotin to protein (MR)

MR = M/P

Where:

  • M = Biotin Concentration
  • P = Protein Concentration

References


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