Glycerol Content Assay kit (Enzymatic, Micro Method) - BioReagent, high purity

Cat. No.: G1505530

GRADE & PURITY BioReagent

Overview

Glycerol is an intermediate product in the metabolism of triglycerides in animal and plant tissues as well as in blood. Triglycerides are hydrolyzed to produce glycerol, which is then further oxidized to provide energy for cellular metabolism. Therefore, the glycerol content serves as a reliable indicator for monitoring triglyceride hydrolysis, offering a more convenient detection method.

Detection Principle: Glycerol is phosphorylated to glycerol-3-phosphate by glycerol kinase in the presence of ATP. Glycerol-3-phosphate is then oxidized by glycerol phosphate oxidase, generating hydrogen peroxide. Under the action of peroxidase, a chromogenic substrate is converted to a quinoneimine dye, which has an absorption peak at 505 nm. The glycerol content is calculated by measuring the absorbance at this wavelength.

Detection Range: 31.25 - 2000 nmol/mL
Sensitivity: 31.25 nmol/mL
Applicable Samples: Animal/plant tissues, cells, serum (plasma), or other liquids

Note: Before formal testing, it is recommended to perform a preliminary test with 2-3 samples expected to have significant differences.

User-Prepared Instruments and Reagents

1.Microplate reader or visible spectrophotometer (capable of measuring absorbance at 505 nm)

2.96-well plate or micro glass cuvettes, adjustable micropipettes and tips, 1.5 mL centrifuge tubes

3.Incubator, ice maker, refrigerated centrifuge

4.Deionized water, PBS, Isopropanol

5.Homogenizer or mortar (for tissue samples)

Experimental Procedure

1. Reagent Preparation

Reagent Name	Reagent Preparation	Notes
Working Reagent Ⅰ	Prepare before use: For 48T, add 7 mL Reagent III; for 96T, add 14 mL Reagent III. Dissolve completely.	Unused reagent can be aliquoted and stored at -20°C protected from light for one month. Avoid repeated freeze-thaw cycles.
Working Reagent Ⅱ	Prepare before use: For 48T, add 7 mL Reagent III; for 96T, add 14 mL Reagent III. Dissolve completely.	Can be stored at 4°C protected from light for one month.
Working Reagent	Prepare before use: Mix Working Reagent I and Working Reagent II in a 1:1 ratio. Prepare freshly for each use.
Reagent Ⅲ	Ready-to-use; equilibrate to room temperature before use.	Store at 4°C.
Standard	Ready-to-use; equilibrate to room temperature before use. (2 μmol/mL Glycerol Standard)	Store at 4°C protected from light.

2. Standard Curve Setup

Using the 2 μmol/mL Glycerol Standard, prepare a dilution series as shown below:

Tube	Standard Volume	Deionized Water Volume (μL)	Concentration (nmol/mL)
Std.1	200μL of 2 µmol/mL Standard	0	2000
Std.2	100μL of Std.1	100	1000
Std.3	100μL of Std.2	100	500
Std.4	100μL of Std.3	100	250
Std.5	100μL of Std.4	100	125
Std.6	100μL of Std.5	100	62.5
Std.7	100μL of Std.6	100	31.25
Blank	0	100	0

Note: The standard curve must be generated with each experiment. Diluted standard solutions are unstable and must be used within 4 hours.

3. Sample Preparation

Note: Fresh samples are recommended. If not used immediately, samples can be stored at -80°C for one month.

3.1 Tissue
Weigh approximately 0.1 g of sample. Rinse the tissue with PBS, blot dry with filter paper. Add 1 mL of isopropanol and homogenize in an ice bath using a homogenizer or mortar. Centrifuge at 10,000 g, 4°C for 10 minutes. Collect the supernatant for assay.

3.2 Cells
Collect 5 million cells into a centrifuge tube. Wash the cells with cold PBS, centrifuge, and discard the supernatant. Add 1 mL of isopropanol. Disrupt the cells by sonication in an ice bath for 5 minutes (power 20% or 200W, pulse 3s on, 7s off, repeat 30 times). Centrifuge at 10,000 g, 4°C for 10 minutes. Collect the supernatant for assay.

3.3 Serum (Plasma) and Other Liquid Samples
Assay directly.

Note: If protein concentration measurement is required, Aladdin's BCA Protein Quantification Kit (B665595) or Ready-to-Use BCA Protein Quantification Kit (R1491648) is recommended. Since isopropanol denatures proteins, prepare a separate sample aliquot using deionized water according to the extraction steps for protein assay.

4. Assay Steps

4.1 Preheat the microplate reader or visible spectrophotometer for at least 30 minutes. Set the wavelength to 505 nm. For spectrophotometers, zero the instrument with deionized water.
4.2 Assay Procedure (perform in a 96-well plate or micro glass cuvette):

Reagent	Blank Well (μL)	Standard Well (μL)	Control Well (μL)	Test Well (μL)
Sample	0	0	0	20
Isopropanol	0	0	20	0
Standard	0	20	0	0
Deionized Water	20	0	0	0
Working Reagent	200	200	200	200

4.3 Mix immediately and record the initial absorbance at 505 nm (A₁). Incubate at 37°C for 10 minutes. Quickly remove and record the absorbance at 10 minutes (A₂). Calculate ΔA = A₂ - A₁ for each well. Record ΔA for the Blank well as ΔA _blank , for the Standard well as ΔA _standard , for the Control well as ΔA _control , and for the Test well as ΔA _test . Calculate ΔΔA _test = ΔA _test - ΔA _control and ΔΔA _standard = ΔA _standard - ΔA _blank .

Note: The Control, Blank, and Standard wells only need to be set up 1-2 times. A preliminary test with 2-3 samples showing expected significant differences is recommended. If ΔΔA _test is less than 0.004, consider increasing the sample volume. If ΔΔA _test is greater than the ΔΔA _standard of the 1000 nmol/mL standard, further dilute the sample with isopropanol (multiply the result by the dilution factor) or reduce the sample amount used for extraction.

5. Calculation of Results

Note: We provide both derived and simplified calculation formulas. They are equivalent. The simplified formulas in bold are recommended for final calculation.

5.1 Standard Curve Plotting
Plot the standard concentration (x-axis) against ΔΔA_standard (y-axis) to generate the standard curve and obtain the standard equation. Substitute ΔΔA_test into the equation to obtain x (nmol/mL).

5.2 Glycerol Content Calculation

(1) Based on Protein Concentration

Glycerol (nmol/mg protein) = (V_sample × x) ÷ (V_sample × Cpr)= x ÷ Cpr

(2) Based on Sample Fresh Weight

Glycerol (nmol/g fresh weight) = (V_sample × x) ÷ (W × V_sample ÷ V_{total sample})= x ÷ W

(3) Based on Cell Count

Glycerol (nmol/10⁶ cells) = (V_sample × x) ÷ (n × V_sample ÷ V_{total sample})= x ÷ n

(4) Based on Liquid Volume
Glycerol (nmol/mL) = (V_sample × x) ÷ V_sample= x

Parameter Definitions:

• V_sample: Volume of sample added to the reaction system (0.02 mL)

• V_{total sample}: Volume of isopropanol added during extraction (1 mL)

• Cpr: Sample protein concentration (mg/mL)

• W: Sample weight (g)

• n: Number of cells (in units of 10⁶)

6. Representative Results

The following data is for reference only. The experimenter needs to perform assays based on their specific samples.

Glycerol Standard Curve: y = 0.0003x - 0.0018, R² = 1

                                  Figure 1: Glycerol Standard Curve

Precautions

1. Before formal testing, it is recommended to perform a preliminary test with 2-3 samples expected to have significant differences.

2. This product is for research use only. Not for use in clinical diagnosis. For your safety and health, please wear a lab coat and disposable gloves during operation.

Storage and Shipping

Storage

Store at 2-8°C,Protected from light,Store at -20°C

Shipped In

Ice chest + Ice pads

Stability And Storage

Each component has a shelf life of 6 months under corresponding storage conditions. G1505530A: Store at -20℃ long term (6 months). Store in the dark. G1505530B, G1505530D: Store at 2-8℃ long term (6 months). Store in the dark. G1505530C: Store at 2-8℃ lon

Contents & Storage

G1505530	Component	48 T	96 T	Storage
G1505530A	Reagent Ⅰ	1EA (48T)	1EA (96T)	-20℃. Store in the dark.
G1505530B	Reagent Ⅱ	1EA (48T)	1EA (96T)	2-8℃. Store in the dark.
G1505530C	Reagent Ⅲ	15 mL	30 mL	2-8℃.
G1505530D	Standard	1 mL	1 mL	2-8℃. Store in the dark.