Triglyceride Content Assay Kit (Spectrophotometry)

Triglycerides (TG) are extracted with isopropanol. After saponification of TG with KOH, they are hydrolyzed to produce glycerol and fatty acids. Glycerol is then oxidized by periodate to generate formaldehyde. In the presence of chloride ions, formaldehyde condenses with acetylacetone to form a yellow compound, which has a characteristic absorption peak at 420 nm. The color intensity is proportional to the TG content.

Taking 50T/48S packing for example:

I. Required Equipment & Materials:

Visible spectrophotometer/microplate reader, micro glass cuvettes/96-well plates, water bath, adjustable pipettes, double-distilled water, n-heptane, isopropanol, and glass vials.

II. Extraction of TG:

1.Extraction of TG from tissues:

Homogenize the tissue in an ice bath at a ratio of tissue weight (g) to CB0117S-A volume (mL) of 1:5–10 (it is recommended to weigh ~0.1 g tissue and add 1 mL of CB0117S-A). Centrifuge at 8000 × g at 4 °C for 10 min. Collect the supernatant as the TG test solution.

2.Extraction of TG from cells or bacteria:

Collect 4–5 million cells or bacteria into a centrifuge tube and discard the supernatant. Add 1 mL of CB0117S-A and disrupt by ultrasonication for 1 min (20% intensity, 2 s on, 1 s off). Centrifuge at 8000 × g at 4 °C for 10 min. Collect the supernatant as the TG test solution.

3.Serum (plasma) samples:

Measure directly.

III. Assay Procedure

1.Preheat the visible spectrophotometer/microplate reader for 30 minutes, set the wavelength to 420 nm, and zero with distilled water.

2.Preheat the water bath to 65°C.

3.Perform the following steps in EP tubes according to the table below:

4.Determination of triglyceride content:

Note: The blank and standard tubes only need to be measured 1–2 times.

IV. Calculation

1.Calculation of triglyceride (TG) content in serum (plasma):

TG content (mg/dL) = C_standard × (A_sample − A_blank) ÷ (A_standard − A_blank) × 100 = 100 × (A_sample − A_blank) ÷ (A_standard − A_blank)

C_standard: 1 mg/mL

100: Unit conversion factor (1 dL = 100 mL)

2.Calculation of triglyceride (TG) content in tissues:

(1) Calculated based on protein concentration

TG content (mg/mg prot) = C_standard × V × (A_sample − A_blank) ÷ (A_standard − A_blank) ÷ (C_pr × V) = (A_sample − A_blank) ÷ (A_standard − A_blank) ÷ C_pr

(2) Calculated based on sample weight

TG content (mg/g) = C_standard × V × (A_sample − A_blank) ÷ (A_standard − A_blank) ÷ W = (A_sample − A_blank) ÷ (A_standard − A_blank) ÷ W

C_standard: 1 mg/mL

C_pr: Protein concentration of the sample (mg/mL)

W: Fresh weight of the sample (g/mL)

V: Volume of Reagent I added (1 mL)

3.Calculation of triglyceride (TG) content in cells or bacteria:

TG content (mg/10⁴ cells) = C_standard × (A_sample − A_blank) ÷ (A_standard − A_blank) ÷ cell or bacterial concentration (10⁴ cells/L) = (A_sample − A_blank) ÷ (A_standard − A_blank) ÷ cell or bacterial concentration (10⁴ cells/L)

C_standard: 1 mg/mL

1.The kit contains volatile substances. Gloves and a mask should be worn during the experiment. After opening, reagent bottles should be tightly capped immediately.

2.After adding CB0117S-B, vortex vigorously to ensure thorough extraction of triglycerides from the sample. The shaking amplitude, duration, number of repetitions, and phase separation time should be kept consistent.

3.To ensure reproducibility, the cooling time after each water bath should be kept consistent.

4.If the OD value of the test sample exceeds 1.0, it is recommended to appropriately dilute the sample with CB0117S-A before measurement, and multiply by the corresponding dilution factor in the final calculation.

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

6.Please wear a lab coat and disposable gloves.

• CB0117S-Instruction Manual(250 KB)