Fatty Acid Synthase Assay Kit (Microanalysis)

Fatty acid synthase catalyzes the conversion of acetyl-CoA, malonyl-CoA, and NADPH to long-chain fatty acids and NADP⁺. NADPH exhibits an absorption peak at 340 nm, whereas NADP⁺ does not. FAS activity is calculated by measuring the rate of decrease in absorbance at 340 nm.

Taking 100T/96S packing for example:

I. Required Equipment & Materials:

Mortar, ice, benchtop centrifuge, UV spectrophotometer, micro quartz cuvette/96-well plate, adjustable pipettes, and distilled water.

II. Crude Enzyme Extraction:

1.Tissue:

According to tissue weight (g), add CB0071M-A at a ratio of 1:5–10 (mL) (recommended: weigh about 0.1 g tissue and add 1 mL CB0071M-A), then homogenize in an ice bath. Centrifuge at 16,000 rpm for 40 min at 4°C, collect the supernatant, and keep it on ice for analysis.

2.Bacteria or fungi:

According to the number of cells (10⁴), add CB0071M-A at a ratio of 500–1000:1 (mL) (recommended: add 1 mL CB0071M-A to 5 × 10⁶ cells). Disrupt the cells by ultrasonication in an ice bath (power 300 W, sonicate for 3 s with 7 s intervals, total time 3 min). Then centrifuge at 16000 rpm for 40 min at 4°C, collect the supernatant, and keep it on ice for analysis.

3.Serum or other liquid samples:

Measure directly.

III. Assay Procedure

1.Preheat the spectrophotometer for at least 30 minutes, set the wavelength to 340 nm, and use distilled water to zero the instrument.

2.Preheat CB0071M-D in a 40°C water bath for 30 min.

3.Sample measurement (add the following reagents sequentially into a 1 mL quartz cuvette):

IV. Calculation of FAS Activity

1. When using a micro quartz cuvette for measurement, the calculation formula is as follows:

(1) Based on protein concentration:

Definition: One unit of enzyme activity is defined as the amount of enzyme that oxidizes 1 μmol NADPH per minute per mg protein at 37°C.

FAS (μmol/min/mg prot) = [(ΔA_sample − ΔA_blank) ÷ ε ÷ d × V_total × 10⁶] ÷ (Cpr × V_sample) ÷ T = 1.61 × (ΔA_sample − ΔA_blank) ÷ Cpr

(2) Based on sample mass:

Definition: One unit of enzyme activity is defined as the amount of enzyme that oxidizes 1 μmol NADPH per minute per g tissue at 37°C.

FAS (μmol/min/g) = [(ΔA_sample − ΔA_blank) ÷ ε ÷ d × V_total × 10⁶] ÷ (W × V_sample ÷ V_total_sample) ÷ T = 1.61 × (ΔA_sample − ΔA_blank) ÷ W

(3) Based on cell number:

Definition: One unit of enzyme activity is defined as the amount of enzyme that oxidizes 1 μmol NADPH per minute per 10⁴ cells at 37°C.

FAS (μmol/min/10⁴ cell) = [(ΔA_sample − ΔA_blank) ÷ ε ÷ d × V_total × 10⁶] ÷ (cell number × V_sample ÷ V_total_sample) ÷ T = 1.61 × (ΔA_sample − ΔA_blank) ÷ cell number

(4) Based on liquid volume:

Definition: One unit of enzyme activity is defined as the amount of enzyme that oxidizes 1 μmol NADPH per minute per mL sample at 37°C.

FAS (μmol/min/mL) = [(ΔA_sample − ΔA_blank) ÷ ε ÷ d × V_total × 10⁶] ÷ V_sample ÷ T = 1.61 × (ΔA_sample − ΔA_blank)

Note:

ε: Molar extinction coefficient of NADPH, 6.22 × 10³ L/mol/cm

d: Optical path length of cuvette, 1 cm

V_total: Total reaction volume, 200 μL = 2 × 10⁻⁴ L

Cpr: Protein concentration of the supernatant (mg/mL)

W: Sample weight

V_sample: Volume of supernatant added to the reaction system, 20 μL = 0.02 mL

V_total_sample: Volume of extraction solution, 1 mL

T: Reaction time, 1 min

2. When using a 96-well plate, the calculation formula is as follows:

(1) Based on protein concentration:

Definition: One unit of enzyme activity is defined as the amount of enzyme that oxidizes 1 μmol NADPH per minute per mg protein at 37°C.

FAS (μmol/min/mg prot) = [(ΔA_sample − ΔA_blank) ÷ ε ÷ d × V_total × 10⁶] ÷ (Cpr × V_sample) ÷ T = 3.22 × (ΔA_sample − ΔA_blank) ÷ Cpr

(2) Based on sample mass:

Definition: One unit of enzyme activity is defined as the amount of enzyme that oxidizes 1 μmol NADPH per minute per g tissue at 37°C.

FAS (μmol/min/g) = [(ΔA_sample − ΔA_blank) ÷ ε ÷ d × V_total × 10⁶] ÷ (W × V_sample ÷ V_total_sample) ÷ T = 3.22 × (ΔA_sample − ΔA_blank) ÷ W

(3) Based on cell number:

Definition: One unit of enzyme activity is defined as the amount of enzyme that oxidizes 1 μmol NADPH per minute per 10⁴ cells at 37°C.

FAS (μmol/min/10⁴ cell) = [(ΔA_sample − ΔA_blank) ÷ ε ÷ d × V_total × 10⁶] ÷ (cell number × V_sample ÷ V_total_sample) ÷ T = 3.22 × (ΔA_sample − ΔA_blank) ÷ cell number

(4) Based on liquid volume:

Definition: One unit of enzyme activity is defined as the amount of enzyme that oxidizes 1 μmol NADPH per minute per mL sample at 37°C.

FAS (μmol/min/mL) = [(ΔA_sample − ΔA_blank) ÷ ε ÷ d × V_total × 10⁶] ÷ V_sample ÷ T = 3.22 × (ΔA_sample − ΔA_blank)

Note:

ε: Molar extinction coefficient of NADPH, 6.22 × 10³ L/mol/cm

d: Optical path length of 96-well plate, 0.5 cm

V_total: Total reaction volume, 200 μL = 2 × 10⁻⁴ L

Cpr: Protein concentration of the supernatant (mg/mL)

W: Sample weight

V_sample: Volume of supernatant added to the reaction system, 20 μL = 0.02 mL

V_total_sample: Volume of extraction solution, 1 mL

T: Reaction time, 1 min

1.The prepared reagents should be stored at 4°C and used within 3 days.

2.For protein concentration determination, it is recommended to use the BCA Protein Quantification Kit (C0050) produced by TargetMol.

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

4.Please wear a lab coat and disposable gloves.

• CB0071M-Instruction Manual(311 KB)