Fructose 1,6-bisphosphatase (FBP) kit instructions

Fructose 1,6-bisphosphateFructose 1,6-bisphosphatase, FBP ) Kit Instructions

Micro method 100 tubes/96 samples

NoteMean :Be sure to take 2-3 samples with large expected differences for pre-determination before the formal measurement

Measurement significance:

Fructose-1,6-bisphosphatase, also known as fructose-1,6-bisphosphatase, catalyzes fructose-1,6-bisphosphate and water to generate fructose-6-phosphate and inorganic phosphorus. Plays a key role in the synthesis of the assimilator sucrose.

Determination principle:

 FBP catalyzes fructose-1,6-diphosphate and water to fructose-6-phosphatet and inorganic phosphorus, and the added phosphate glucose isomerase and 6-phosphate glucose dehydrogenase in the reaction system in turn catalyze the formation of 6-phosphogluconic acid and NADPH, FBP activity can be calculated by measuring the rate of increase of NADPH at 340 nm.

Needhome-growntools and supplies :

Spectrophotometer/microplate reader, constant temperature water bath, benchtop centrifuge, adjustable pipette, microquartz cuvette/96-well plate, mortar, ice and distilled water.

Composition and preparation of reagents: >

Extraction one:Liquid 100mL×1 bottle, store at 4°C.

Extract 2: Liquid 100mL×1 bottle, store at 4°C.

Reagent 1: powder × 1 bottle, store at -20°C; add 20 ml of reagent 4 to complete dig to be dissolved before use, store unused reagent at 4°C;

Reagent 2 : 7.2 μL×1 liquid bottle, store at 4°C; add 1 ml of distilled water to dissolve completely before use, and store unused reagents at 4°C;

Reagent 3: Powder x 1 bottle, -20°C Store; add 1 ml of distilled water to dissolve completely before use, store unused reagents at 4 °C;

Reagent 4: liquid 25 ml × 1 bottle, store at 4 °C;

Pre-treatment of samples:

①Total FBP enzyme extraction: It is recommended to about 0.1 g of sample, add 1 ml of extract solution 1, homogenize in ice bath, then break ultrasonic (ice bath, 200 W, pause for 3 seconds, pause for 7 seconds, total time 1 minute), then centrifuge at 8000 g for 10 minutes at 4°C, and take the supernatant for determination.

② Separation of cytoplasmic and chloroplast FBP enzymes: according tothe ratio of plant tissue mass (g): extract volume (ml) of 1:5-10 (it is recommended to weigh about 0.1 g of sample and add 1 ml of extract solution 1), after homogenization in an ice bath, centrifuge at 4 ° C, 200 g for 5 minutes, discard the precipitate, take the supernatant and centrifuge at 4 °C, 8000 g for 10 minutes, take the supernatant for the determination of cytoplasmic FBPase activity, take the precipitate and add 1 ml of extract 2. After oscillation and dissolution, ultrasonic break (ice bath, 200 W, break for 3 seconds, intermittent 7 seconds, total time 1 minute), then centrifuge at 8000 g for 10 minutes at 4 °C, and take the supernatant to remove the FBPase activity in chloroplast.

It is recommended to determine the total FBP enzyme activity and extract the crude enzyme solution according to step ①. If FBP in the cytoplasm and chloroplast needs to be measured separately, follow step ② to extract the crude enzymesolution.

Determination steps:

 1. Preheat the spectrophotometer or microplate reader for more than 30 minutes, set the wavelength to 340 nm and zero with distilled water.

2. Preheat Reagents 1, 2, and 3 to 37°C (mammals) or 25°C (other species) for 10 minutes.

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