Plant Proline Detection Kit

January 15, 2024

Plant Proline Test Kit ( Ninetrione Colorimetric Method )
Plant Proline Assay Kit with Ninhydrin
● Product composition:
Component number name specification Storage
PP5040-01 Reagent 1-Pro Lysate 100 ml RT
PP5040-02 Reagent 2-Pro standard solution 1 ml 4 ° C
PP5040-03 Reagent 3-Pro color developing solution 50 ml RT protected from light
● Product introduction:
Proline (Pro) is a cyclic α-sub-amino acid which is neutral with an isoelectric point of 6.30 and is readily soluble in water. Under normal environmental conditions, the content of proline is low, but under adverse conditions (dry, hot, cold, frozen, saline, etc.), proline often accumulates, that is, the accumulation index is related to the stress resistance of plants. The detection principle of the plant proline detection kit (ninhydrin colorimetric method) is that the proline reacts with the ninhydrin to generate a stable red product, and the absorbance is measured by a spectrophotometer, and the color is within a certain range. The depth (ie absorbance) is proportional to the concentration of proline.
The kit does not use traditional sulfosalicylic acid and toluene and other harmful solvents, and is economical and environmentally friendly.
The kit is mainly used to determine the free proline content in plant tissues, and is not suitable for the determination of proline in animal and medical samples.
The kit can be used 50 times, using a 1 ml Pro color solution.
● Bring your own materials:
Plant roots, leaves, etc.; scissors; screw cap centrifuge tube or lock cap centrifuge tube or 96-well plate; 1 ml glass cuvette; spectrophotometer or microplate reader; water bath or dry bath; centrifuge
● Operation steps:
1 , sample processing:
Take fresh plant tissue, clean it, dry it, cut it, and quickly weigh it. Add Pro lysate according to the ratio of 20mg weight to 1ml Pro lysate (recommended to use 1.5ml screw centrifuge tube or lock cap centrifuge tube), boiling water bath Or dry bath treatment for 20 minutes (during intermittent mixing), centrifugation at 10,000 rpm for 2 minutes, the supernatant is the proline extract, stored at 4 ° C for use. If the extract to be tested cannot be measured in time, it should be stored at -20 °C and stabilized within 1-2 days.
2 , Pro color solution preparation:
The ninhydrin powder contained in the small tube is all added to the ninhydrin lysate and vigorously mixed until completely dissolved, that is, formulated into a Pro color developing solution. Note: Pro coloring solution is corrosive, please be careful.
3, the preparation of proline standard solution series:
Take 1 ml of the reagent 1-Pro lysate and add it to the small tube containing the Pro standard solution, mix and dissolve completely, and prepare the Pro standard solution (100 mg/ml).
Add 1 μl of 100 mg/ml standard solution to 1 ml of Pro lysate, and prepare 100 μg/ml Pro standard solution for use. Refer to the table below to prepare different concentrations of Pro standard solution No. 1-6.
Numbering 1 2 3 4 5 6 7 8
100μg/ml Pro standard solution addition amount (ml) 0.01 0.02 0.04 0.06 0.08 0.10 0.15 0.2
Pro Lysate (ml) 0.49 0.48 0.46 0.44 0.42 0.4 0.35 0.3
Proline content (μg) 1 2 4 6 8 10 15 20
4 , sample determination :
= 1 \* GB3 1 Spectrophotometer determination: Set the reaction mixture according to the following table, and add reagents in sequence:
Blank tube Standard tube Measuring tube
Reagent 1-Pro Lysate 0.5 ml - -
Proline extract in step 1 - - 0.5 ml
Pro standard solution (1-8 tubes in step 3) - 0.5 ml -
Reagent 3-Pro color developing solution 1 ml 1 ml 1 ml
= 2 \* GB3 2 Determination of the microplate reader: Set the reaction mixture according to the following table, and add the reagents in sequence:
Blank tube Standard tube Measuring tube
Reagent 1-Pro Lysate 50 μl - -
Proline extract in step 1 - - 50 μl
Pro standard solution (1-8 tubes in step 3) - 50 μl -
Reagent 3-Pro color developing solution 100 μl 100 μl 100 μl
3 Boil or dry in a water bath for 20 minutes, mix intermittently. Care must be taken to avoid liquid spillage when heating. Use a capped centrifuge tube or a screw cap centrifuge tube.
4 Cool in ice bath or running water to room temperature, centrifuge at 10,000 rpm for 2 minutes. If no insoluble matter is found, it may not be centrifuged.
5 Take the supernatant, use a blank tube as a control, and measure the absorbance at 520 nm of the reaction mixture by spectrophotometer or microplate reader.
5 , calculation:
The standard proline standard (1-6 tube) content (μg) was plotted on the abscissa, and the corresponding absorbance was plotted on the ordinate. A standard curve was prepared, and the proline content was calculated according to the absorbance of the tube.


Proline content of plant tissue samples (μg/g) = (C × V T ) / (W × V S )
C = proline content (μg) found on the standard curve
V T = total volume of proline extract in step 1 (ml)
W = fresh weight of sample used in step 1 (g)
V S = volume of proline extract added during the measurement in step 4 (ml)

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