Tissue Inorganic Phosphorus Content Assay Kit (MB, Micro Method)

Cat. No.: O1506749

GRADE & PURITY BioReagent

Overview

  Inorganic phosphorus primarily refers to inorganic phosphate ions, which participate in various metabolic processes within organisms, including energy metabolism, nucleic acid metabolism, and protein phosphorylation/dephosphorylation. Furthermore, it promotes the synthesis, transformation, and transport of carbohydrates.

  Assay Principle:Ammonium molybdate reacts with inorganic phosphate ions to form a complex with a characteristic absorption peak at 660 nm. The inorganic phosphorus content is calculated by measuring the absorbance at 660 nm.

  Applicable Samples: Animal and plant tissues

O1506749	Component	96T	Storage
O1506749A	Reagent I	120 mL	2-8℃
O1506749B	Reagent II	3 mL	2-8℃
O1506749C	Reagent III	1 EA	2-8℃. Store in the dark.
O1506749D	Reagent IV	1 EA	2-8℃. Store in the dark.
O1506749E	Standard	1 mL	2-8℃

Note: It is recommended to perform a pilot experiment with 2-3 samples expected to have significant differences before formal testing.

Required Materials Not Provided

1. Microplate reader or visible spectrophotometer (capable of measuring absorbance at 660 nm)

2. 96-well plate or micro glass cuvettes, Adjustable pipettes and tips

3. Adjustable water bath, Benchtop centrifuge

4. Deionized water, Homogenizer

Experimental Procedure

1. Reagent Preparation

Reagent Name	Preparation Steps	Notes & Storage
Reagent I	Ready-to-use; equilibrate to room temperature before use.	Store at 4°C. Pungent odor - recommended to use in a fume hood.
Reagent II	Ready-to-use; equilibrate to room temperature before use.	Store at 4°C. Corrosive - recommended to use in a fume hood.
Reagent III	Before use, dissolve the vial contents in 1.5 mL deionized water (incubation in a 37°C water bath for 5 min may aid dissolution).	Stable at 4°C protected from light for 1 week.
Reagent IV	Before use, dissolve the vial contents in 6 mL deionized water.	Stable at 4°C protected from light for 1 week.
Phosphorus Assay Reagent	Prepare by mixing Deionized Water : Reagent II : Reagent III : Reagent IV at a ratio of 10 : 5 : 2 : 8.	Prepare according to actual need, use on the same day.
Standard	Ready-to-use; equilibrate to room temperature before use.	Store at 4°C.

2. Sample Preparation

• Tissue Samples: Weigh approximately 0.1 g of tissue.

• Add 1 mL of Reagent I (using a ratio of tissue mass (g) : Reagent I volume (mL) = 1:10).

• Homogenize in an ice bath.

• Centrifuge at 10,000 g, 4°C for 10 minutes.

• Collect the supernatant and keep on ice for assay.

Note: If determining protein concentration is required, the use of the Aladdin B665595 BCA Protein Quantification Kit or R1491648 Ready-to-Use BCA Protein Quantification Kit is recommended.

3. Assay Procedure

3.1 Instrument Preparation

• Preheat the microplate reader or visible spectrophotometer for 30 minutes. Set wavelength to 660 nm.

• Zero the visible spectrophotometer with deionized water.

3.2 Assay Setup (Perform in 1.5 mL centrifuge tubes):

Reagent (μL)	Blank Tube	Standard Tube	Test Tube
Sample Supernatant	0	0	10
Deionized Water	100	90	90
Standard	0	10	0
Phosphorus Assay Reagent	100	100	100

3.3 Measurement

Mix well after addition.

Incubate in a 40°C water bath for 10 minutes.

Cool at room temperature for 10 minutes.

Transfer 200 μL of the supernatant to a micro glass cuvette or 96-well plate.

Measure the absorbance at 660 nm.

Record the values as A_Blank, A_Standard, and A_Test. Calculate ΔA_test = A_Test - A_Blank and ΔA_standard = A_Standard - A_Blank. 

Note: The Blank and Standard tubes need only be measured once. 

A pilot experiment with 2-3 variable samples is recommended. 

If ΔA_test is too small, consider increasing the sample amount. 

If ΔA_test is greater than 1.0, dilute the sample further with Reagent I and multiply the result by the dilution factor, or reduce the sample amount used for extraction.

4. Calculation of Results

Note: We provide both the derived formula and the simplified formula for your convenience. They are mathematically equivalent. The bolded simplified formula is recommended for final calculations.

4.1 Based on Protein Content:

  Derived Formula: Inorganic Phosphorus Content (μmol/mg prot) =(C_Standard × ΔA_test ÷ ΔA_standard) × V_total ÷ C_pr

  Simplified Formula: Inorganic Phosphorus Content (μmol/mg prot) = 1 × ΔA_test ÷ ΔA_standard ÷ C_pr

4.2 Based on Sample Mass:

Derived Formula: Inorganic Phosphorus Content (μmol/g fresh weight) = (C_Standard × ΔA_test ÷ ΔA_standard) × V_total ÷ W 

Simplified Formula: Inorganic Phosphorus Content (μmol/g fresh weight) = 1 × ΔA_test ÷ ΔA_standard ÷ W

Parameter Definitions:

C_Standard: 

Standard solution concentration, 1 mmol/L 

V_total: Total volume of the supernatant, 1 mL = 0.001 L 

W: Sample mass, g 

C_pr: Sample protein concentration, mg/mL

5. Results Example

5.1 Pothos Plant Sample:

Sample: 0.1 g Pothos mixed with 1 mL Reagent I, centrifuged. 

Measured: A_Test=0.334, A_Blank=0.043, A_Standard=0.311 

ΔA_test = 0.334 - 0.043 = 0.291; ΔA_standard = 0.311 - 0.043 = 0.268 

Inorganic Phosphorus Content (μmol/g sample) = 1 × 0.291 / 0.268 / 0.1 = 10.86 μmol/g sample

5.2 Mouse Muscle Sample:

Sample: 0.04 g mouse muscle mixed with 0.4 mL Reagent I, centrifuged. 

 Measured: A_Test=1.201, A_Blank=0.042, A_Standard=0.29 ΔA_test = 1.201 - 0.042 = 1.159; ΔA_standard = 0.29 - 0.042 = 0.248 

 Inorganic Phosphorus Content (μmol/g sample) = 0.4 × 1.159 / 0.248 / 0.04 = 46.73 μmol/g sample 

 Note: Here V_total is 0.4 mL, not 1 mL, as per the sample preparation described.

Precautions

1.The Phosphorus Assay Reagent must be prepared fresh before use and used on the same day. It is recommended to complete the measurements within 40 minutes.

2.This product is for research use only. Not for use in diagnostic procedures. For your safety and health, please wear lab coats and disposable gloves during operation.

Storage and Shipping

Storage

Store at 2-8°C,Protected from light

Shipped In

Wet ice

Stability And Storage

Store at 2-8℃ long term (12 months). Store in the dark.