Application

This kit can be used to measure sucrose content in plant tissue samples. 

Detection significance

Sucrose is a disaccharide which composed by glucose and fructose and it is the main nutrient in most plant cells. The biosynthesis of sucrose depends on the catalysis of sucrose phosphate synthase and 6’-sucrose phosphate phosphatase. It can participate in glycolysis and tricarboxylic acid cycle to produce ATP and NADH.

Detection principle

Sucrose in plant tissue is hydrolyzed to glucose and fructose in boiling water bath under acidic conditions. 5-hydroxymethyl furfural was synthesized from fructose under acid condition and measure the ultraviolet absorption of 5-hydroxymethyl furfural. Glucose must be dissimilated into ketose structure and reduced to obtain 5-hydroxymethylfurfural, but the rate of isomerization of glucose to ketose is very slow. Therefore, the ultraviolet absorption of glucose is much smaller than fructose.

Experimental instrument

Micropipette, Vortex mixer, Centrifuge, Spectrophotometer (290 nm)，temperature water bath

Sample preparation

10% tissue homogenate: Weigh the tissue accurately. Add 9 times the volume of PBS (0.01 M, pH 7.4) according to the proportion of Weight (g): Volume (mL) =1:9. Mechanical homogenate the sample in ice water bath. Centrifuge at 3500 rpm for 15 min. Take the supernatant for detection. Measure the protein concentration of the homogenate with BCA method at the same time.

Operation steps

It is recommended to take 2~3 samples which expected large difference to do pre-experiment before formal experiment.

1. Blank tube: add 0.03 mL of distilled water into a 5 mL glass tube.

Standard tube: add 0.03 mL of 20 μmol/mL sucrose standard into a 5 mL glass tube.

Sample tube: add 0.03 mL of Sample into a 5 mL glass tube.  

2. Add 2.0 mL of Reagent 1 and mix fully with vortex mixer.

3. Tighten the tubes with preservative film and make a hole on the film. Incubate the tubes in 100℃ water bath for 8 min. Cool the tubes with running water.

4. Set the spectrophotometer to zero with double-distilled water and measure the OD values of each tube at 290 nm with 1 cm diameter cuvette.

Note: It can be refer to the following operating table.

Technical parameters

1. The sensitivity of the kit is 0.32 μmol/mL.

2. The intra-assay CV is 3.4% and the inter-assay CV is 8.5%.

3. The recovery of the kit is 102%.

4. The linear range of the kit is 0.32~70 μmol/mL.

Notes

1. The kit is for scientific research only.

2. Instructions should be followed strictly, changes of operation may result in unreliable results.

3. The valid of kit is 6 months.

4. Do not use components from different batches of kit.