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

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

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.

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.

1. Prepare the following controls and samples as outlined in the table below:

   Positive Control	Negative Control	Test Sample*
   d-Biotin (Component C): 2 µL	ddH2O: 2 µL	2 µL
   HABA/Avidin working solution: 18 µL	HABA/Avidin working solution: 18 µL	HABA/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.

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

M = [ΔA₅₀₀ ÷ ε_HABA/Biotin] x DF

Where:

• ε_HABA/Biotin = 34,500 M^-1cm^-1
• DF = Dilution Factor

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

MR = M/P

Where:

• M = Biotin Concentration
• P = Protein Concentration