Solarbio Tissue Total Phosphorus Content Assay Kit

Tissue Total Phosphorus Content Assay Kit

Note: Take two or three different samples for prediction before test.

Detection instrument: Spectrophotometer /Microplate reader

Cat No: BC2855

Size: 100T/96S

Components:

Reagent I: 20 mL×1. Storage at 4℃ (strong corrosive, strong oxidizing). Reagent Ⅱ: 7 mL×1. Storage at 4℃.

Reagent III: Powder×2. Storage at 4℃ and protected from light. Working solution: Fully dissolved with 5 mL of distilled water, then add 2.5 mL of Regent II, mix well.

Standard: 1 mL×1, 1 mmol/L inorganic phosphorus standard. Storage at 4℃.

Product Description:

The form of phosphorus includes inorganic phosphorus and organic phosphorus. Inorganic phosphorus mainly refers to phosphate radical, which is involved in many kinds of metabolism, including energy metabolism, nucleic acid metabolism, protein phosphorylation, dephosphorylation, and so on. By measuring the content of total phosphorus and inorganic phosphorus, the utilization rate of phosphorus in crops can be understood, and the basis for rational fertilization can be provided.

After digestion, total phosphorus was converted into inorganic phosphorus. The molybdenum blue method is a classical method for determining the content of inorganic phosphorus. Under certain conditions, molybdenum blue and phosphate from a substance with a characteristic absorption peak at 660 nm. By measuring the light absorption of 660 nm, the inorganic phosphorus content can be calculated, and then the total phosphorus content in the tissue can be calculated.

Required material

Centrifuge, spectrophotometer/microplate reader, water bath, micro glass cuvette/96 well flat-bottom plate, transfer pipette, distilled water, concentrated sulfuric acid.

Procedure:

I.  Sample Extraction:

0.1g of sample with 1mL of concentrated sulfuric acid (cover tightly to prevent moisture loss) put in boiling water bath for 10 minutes. When the solution is black or brown, take it out. Add 200μL of Reagent I after cooling, mix well. Continue boiling (cover tightly) until the solution is transparent, then cool to room temperature, add 3.8mL of distilled water and mix well. Centrifugated at, 10000rpm and room temperature for 10 minutes, supernatant is used for test.

II. Determination procedure:

1. Preheat the spectrophotometer/ microplate reader for 30 min, adjust wavelength to 660 nm, set zero with distilled water.
2. Set the temperature of water bath to 40℃.
3. Add reagents with the following list:

Mix well, 40℃ water bath for 10 minutes, detect the absorbance at 660 nm after cooling at room temperature for 10 minutes. Record as AB, AS  and AT respectively.

Calculation:

Total phosphate (mmol/g fresh weight)=[C×(AT- AB)÷(AS  – AB)]×V÷W

=0.005×(AT  – AB)÷(AS  – AB)÷W

C: standard concentration, 1mmol/L; V: supernatant volume, 5 mL=0.005 L; W: Sample weight, g.

Note:

1．Reagent III should be prepared before use and only be used the same day.

2．Before the determination, 1–2 samples shall be used for pre-test. If the absorption value is greater than 1.5, distilled water shall be used for corresponding dilution.

Experimental example:

1. Take 0.1g kidney according to the extraction procedure, centrifugally take it up and clean it, and then follow the measurement procedure. Use 96 well plate to calculate: AT= 0.191, AB= 0.051, AS= 0.282. Calculate the total phosphorus content according to the sample mass

Total phosphorus content (mmol/g mass) = 0.005 × (A-AB) ÷ (AS-AB) ÷ W= 0.005×(0.191-0.051) ÷ (0.282-0.051) ÷ 0.1= 0.030 mmol/g mass.

2. Take 0.1g spleen according to the extraction procedure, centrifugally take the cleaning, and then follow the measurement procedure. Use 96 well plate to calculate: AT= 0.197, AB= 0.051, AS= 0.282, calculate the total phosphorus content according to the sample mass

Total phosphorus content (mmol/g mass) =0.005×(A-AB) ÷(AS-AB)÷W= 0.005 × (0.197-0.051) ÷ (0.282- 0.051) ÷ 0.1= 0.032 mmol/g mass.

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Technical Specifications:

The detection limit: 0.0338 mmol/L Linear range: 0.625-8 mmol/L