VB1 Assay Kit (Spectrophotometry)

Vitamin B1 reduces potassium ferricyanide to potassium ferrocyanide under alkaline conditions. The resulting potassium ferrocyanide reacts with Fe³⁺ under weakly acidic conditions to form Prussian blue, which exhibits a characteristic absorption peak at 704 nm.

Taking 50T/48S for example:

Prior to the formal determination, a preliminary assay should be conducted using 2-3 samples with large expected differences.

1. Preparation of Lab Instruments

Visible spectrophotometer, 1 mL glass cuvette, constant-temperature water bath, balance, mortar, centrifuge, and distilled water.

2. Crude Enzyme Extract Preparation

1)Tissue samples:

Grind the sample thoroughly. Add extraction buffer at a ratio of tissue weight (g) to extraction volume (mL) of 1:5–10 (recommended: weigh ~0.1 g tissue and add 0.6 mL CB0190S-A). Incubate at 60 °C for 30 min, then add 0.4 mL distilled water and mix well. Centrifuge at 13,000 × g, 25 °C for 10 min, and collect the supernatant for analysis.

For samples with high protein content (e.g., animal tissues), centrifugation for 20–30 min is recommended.

2)Cell samples:

Add extraction buffer at a ratio of 500–1000 cells (×10⁴) per 1 mL extraction buffer (recommended: 5 × 10⁶ cells + 0.6 mL CB0190S-A). Lyse cells by ultrasonic disruption on ice (power 300 W; sonication 3 s on / 7 s off, total time 3 min). Add 0.4 mL distilled water, mix well, then centrifuge at 13,000 × g, 25 °C for 10 min. Collect the supernatant for analysis.

Serum and other liquid samples: Measure directly.

3. Measurement Procedure

Preheat the spectrophotometer for 30 min, set the wavelength to 704 nm, and zero the instrument using distilled water.

Add the following reagents sequentially into an EP tube:

4. Calculation of VB1

Standard curve:

x-axis: The concentrations of the standard solutions

y-axis (with A blank as the zero point of the standard): The corresponding A standard

y = kx + b. A sample is then substituted into the equation to calculate x (mg/mL).

1)Based on protein concentration

VB1 content (mg/mg prot) = x×V2÷（V2×Cpr）= x÷Cpr

2)Based on sample mass

VB1 content (mg/g) = x×V2÷W=0.6x÷W

3)Based on cell number

VB1 content (mg/104 cell) = x×V2÷cell number（×10,000）=0.6x÷cell number（×10,000）

4)Based on solution volume

VB1 content (mg/mL) = x×V1÷V1= x

Note:

V1: Volume of sample added, 0.1 mL;

V2: Volume of the sample extraction, 0.6 mL;

Cpr: Protein concentration, mg/mL;

W: Sample weight, g

1.For protein quantification, it is recommended to use BCA Protein Quantification Kit (C0050).

2.If the absorbance value exceeds 1, dilute the sample before measurement and multiply the calculated result by the dilution factor.

3.For samples with high protein concentrations, such as animal tissues, if precipitation occurs after color development, dilute the sample and re-measure, and multiply the result by the dilution factor.

4.Measure the absorbance immediately after color development is complete.

5.The linear range of the standard curve is 0.1–10 μg/mL.

6.The product is for R&D use only, not for diagnostic procedures, food, drug, household or other uses.

7.Please wear a lab coat and disposable gloves.

• CB0190S-Instruction Manual(296 KB)