Plant trehalose-6-phosphate (T6P) ELISA kit

　　Plant trehalose-6-phosphate (T6P) ELISA kitUser Manual

BS-8050-A Plant Trehalose-6-phosphate (T6P) ELISA Kit 96T

BS-8050-B Plant Trehalose-6-phosphate (T6P) ELISA Kit 48T

1、 Experimental principle

This kit uses a double antibody sandwich enzyme-linked immunosorbent assay (ELISA) to detect T6P levels in plant tissues or cells. Microplate pre coated with anti-T6P antibody, T6P binds to solid-phase antibody in the sample and forms a complex by adding horseradish peroxidase (HRP) labeled secondary antibody. Add substrate TMB for color development, TMB is converted to blue under HRP catalysis, and turns yellow after acid termination. The color depth is positively correlated with the concentration of T6P in the sample, and the concentration is calculated by measuring the absorbance at 450nm wavelength.

2、 Composition of reagent kit

Storage conditions for component specifications

Pre packaged microporous plate with 12 holes and 8 strips, sealed and dried at 2-8 ℃

Standard freeze-dried powder dissolved at -20 ℃ and packaged separately

HRP labeling detection antibody liquid, avoid light exposure 2-8 ℃

Chromogenic substrate (TMB) solution A+solution B is prepared on site and kept away from light

Termination solution 2M sulfuric acid at room temperature

Concentrated detergent liquid dissolved and crystallized in a 40 ℃ water bath

Sample Xishi liquid contains stabilizer

Note: There may be differences in the components of different brands, please refer to the specific instructions for details.

3、 Sample processing

1. Sample type

Plant tissue: Weigh 0.1g of tissue, add 1mL of extract to ice bath homogenate, centrifuge to obtain supernatant.

Cells/bacteria: Collect and discard the supernatant after centrifugation, and extract the supernatant by sonicating the cells.

2. Storage conditions

Short term: Store at 2-8 ℃ for ≤ 48 hours.

Long term: Freeze at -20 ℃ after packaging to avoid repeated freeze-thaw cycles.

3. Dilution suggestion

Conventional sample: It is recommended to dilute 5-20 times (such as taking 20 μ L of sample+180 μ L of diluent).

High concentration samples: Pre experiments are required to determine the dilution factor, with a maximum dilution of 100000 times (three-step dilution method).

4、 Operation steps

Standard product preparation

Dissolve the freeze-dried standard in 1mL of diluent, mix well, and let it stand for 10 minutes.

Perform a series of dilutions (such as 8pmol/L → 4pmol/L →... → 0.5pmol/L).

Add sample

Standard well: 50 μ L/well.

Sample well: First add 40 μ L of diluent, then add 10 μ L of sample (total dilution 5-fold).

Blank hole: No sample or secondary antibody added.

Incubation

Incubate at 37 ℃ for 120 minutes, wash the plate 3 times (soak for 1 minute each time).

Color rendering and termination

Add 100 μ L of TMB chromogenic solution to each well and incubate at 37 ℃ in the dark for 15 minutes.

Add 50 μ L of termination solution to terminate the reaction, and measure the OD value within 30 minutes.

5、 Precautions

Sample avoidance: Samples containing sodium azide (NaN3) (inhibiting HRP activity).

Reagent equilibrium: Room temperature equilibrium is required for 20 minutes before use, and the concentrated washing solution needs to be dissolved in a 40 ℃ water bath for crystallization.

Quality control: It is recommended to set up multiple holes, and the standard curve R ² should be ≥ 0.95.

6、 Result analysis

Standard curve: Plot a four parameter equation of OD value versus concentration.

Concentration calculation: Substitute the sample OD value into the curve equation and multiply it by the dilution factor.

Note: The above is for reference only and is not considered as actual data. Experiments must strictly follow the instructions or consult technical teachers.

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