Combined Starch Synthase Activity Detection Kit Micro Method

Combined Starch Synthase Activity Detection Kit Micro Method

Attention: Before formal testing, it is necessary to take2-3Make predictions based on samples with significant expected differences.

Item Number：BC3295

Specifications100T/96S

Product Content:

extract solution：Liquid 100mL x 1 bottle, stored at 4 ℃;

Reagent 1: 30mL x 1 bottle, stored at 4 ℃;

Reagent 2: Powder x 1 bottle, stored at 4 ℃;

Reagent 3: Powder x 1 bottle, stored at -20 ℃;

Reagent 4: Powder x 2 bottles, stored at 4 ℃; Add 5mL of reagent before use and mix thoroughly for later use;

Reagent 5: Powder x 1 bottle, stored at 4 ℃; Add 10mL of reagent before use and mix thoroughly for later use;

Reagent 6: 1 powder, stored at -20 ℃; Add 500 μ L of double distilled water before use, fully dissolve and set aside for later use;

Reagent 7: Liquid 250 μ L x 2 tubes, stored at -20 ℃;

Reagent 8: Liquid 12.5 μ L x 2 tubes; Add 4mL of dissolved reagent 4 to each vial before use.

reaction solutionⅠPreparation:Before use, add 14mL of reagent one to reagent two, slowly heat and gradually increase the temperature to dissolve it. After cooling, add it to reagent three and mix it for dissolution. This can be prepared and tested in two batches.

Product Description:

GBSS (EC 2.4.1.21) exists in a bound state in starch bodies, catalyzing the elongation reaction of starch chains and mainly responsible for the synthesis of linear starch.

GBSS catalyzes the reaction between ADPG and starch primer (glucan), transferring glucose molecules to the starch primer while generating ADP; Further catalyze NADP by sequentially adding pyruvate kinase, hexokinase, and 6-phosphate glucose dehydrogenase to the reaction system⁺Reduced to NADPH, where the amount of NADPH generated is proportional to the amount of ADP generated in the previous reaction. By measuring the increase in NADPH at 340nm, GBSS activity can be calculated.

Equipment and supplies to be provided:

UV spectrophotometer/microplate reader, water bath, desktop centrifuge, adjustable pipette, trace quartz colorimetric dish/96 well plate (UV plate), mortar, ice, and distilled water.

Operation steps:

1、 Preparation of crude enzyme solution

Weigh about 0.1g of tissue and add 1mL of extraction solution, homogenize in an ice bath. 10000g ， Centrifuge at 4 ℃ for 10 minutes, discard the supernatant, add 1mL of extraction solution to the precipitate, mix well, and place on ice for testing.

2、 Measurement steps

1. Preheat the spectrophotometer or enzyme-linked immunosorbent assay (ELISA) reader for at least 30 minutes, adjust the wavelength to 340nm, and zero the distilled water.

2. Add the following reagents in order into the EP tube

Immediately transfer 200 μ L of trace quartz colorimetric dish or 96 well plate after mixing, record the initial absorbance A1 at 340 nm wavelength and the absorbance A2 after 2 minutes, and calculate Δ A=A2-A1.

Note:If there is precipitation in reagent 2, it should be thoroughly dissolved and mixed before adding.

IIIGBSSActivity Calculation

a.The calculation formula for using a trace quartz colorimetric dish is as follows:

1. Calculated based on sample protein concentration:

The definition of unit: One enzyme activity unit is defined as the catalytic production of 1nmol NADPH per minute per mg of tissue protein in a 1mL reaction system.

GBSS activity (U/mg prot)=[Δ A ÷ (ε× d) × V measurement] ÷ (Cpr × V sample ÷ V total reaction × V supernatant) ÷ T=432 × Δ A ÷ Cpr

This method requires self determination of sample protein concentration.

2. Calculated based on the fresh weight of the sample

The definition of unit: One enzyme activity unit is defined as the catalytic production of 1nmol NADPH per minute per gram of tissue in a 1mL reaction system.

GBSS activity (U/g fresh weight)=[Δ A ÷ (ε× d) × V measurement × 10⁹]÷ (W ÷ V extraction x V sample ÷ V reverse total x V supernatant) ÷ T=432 × Δ A ÷ W.

V measurement: measuring volume, 0.26 mL; V sample: Add sample volume, 0.1mL; V anti total: reaction volume, 0.31mL; V supernatant: add supernatant volume, 0.15mL; V extraction: add extraction solution volume, 1 × 10^-3L. ε: NADPH molar extinction coefficient, 6.22 × 10^-3mL/nmoL/cm； d: Colorimetric dish diameter, 1cm; T: reaction time, 2 minutes; Cpr: sample protein concentration, mg/mL； W: Sample quality, g。

b.use96The calculation formula for orifice plate measurement is as follows:

1. Calculated based on sample protein concentration:

The definition of unit: One enzyme activity unit is defined as the catalytic production of 1 nmol NADPH per minute per mg of tissue protein in the reaction system.

GBSS activity (U/mg prot)=[Δ A ÷ (ε× d) × V measurement] ÷ (Cpr × V sample ÷ V total reaction × V supernatant) ÷ T=720 × Δ A ÷ Cpr.

This method requires self determination of sample protein concentration.

2. Calculated based on the fresh weight of the sample

The definition of unit: One enzyme activity unit is defined as the catalytic production of 1 nmol NADPH per minute per gram of tissue in the reaction system.

GBSS activity (U/g fresh weight)=[Δ A ÷ (ε× d) × V measurement] ÷ (W ÷ V extraction × V sample ÷ V reverse total × V supernatant) ÷ T=720 × Δ A ÷ W.

V measurement: measuring volume, 0.26 mL; V sample: Add sample volume, 0.1mL; V anti total: reaction volume, 0.31mL; V supernatant: add supernatant volume, 0.15mL; V extraction: add extraction solution volume, 1 × 10^-3L. ε: NADPH molar extinction coefficient, 6.22 × 10^-3mL/nmoL/cm； d: Colorimetric dish diameter, 1cm; T: reaction time, 2 minutes; Cpr: sample protein concentration, mg/mL； W: Sample quality, g。

Combined Starch Synthase Activity Detection Kit Micro MethodOrdering instructions:

1. The vast majority of products are in stock and can generally be ordered and shipped on the same day.
2. Some non essential products require a 1-2 day advance booking, while overseas futures require a 3-6 week advance booking.
3. Some product prices may change due to factors such as delivery time and batch. If there are any changes, the new price on the day of ordering shall prevail.
4. The daily order deadline is 16:00 sharp, and in some cities it can be until 17:00 sharp. If the deadline is exceeded and the shipment cannot be made on the same day, it will be arranged for the next day.
5. After processing the payment, please notify us by fax, text message, etc. of the receipt and the recipient's address, name, etc.

Transportation instructions:

Ultra low temperature products: Dry ice is added during the transportation of extremely low temperature products. Wrap the product with dry ice, seal it with a foam box, glue the foam box layer by layer with adhesive tape, put it into the box of SOLEBAR, and then deliver it to the cooperative express delivery, which will deliver it safely, quickly, efficiently, and confidently to the customers, ensuring the stability of the product's nature and escorting your experiment.
Low temperature products: During the transportation of low-temperature products, add Solaibao ice packs for transportation. Wrap the product with ice bags in advance, then seal it with foam boxes, seal the foam boxes with adhesive tape, and put it into the boxes of SOLEBAR (the heat preservation effect can last for a week if it is less), and then deliver it to the cooperative express, which can deliver it safely, quickly, efficiently and confidently to the customers, ensuring the stability of the product properties and escorting your experiment.
Normal temperature products: Normal temperature products do not require ice or special packaging during transportation. The products are quickly distributed by the company's warehouse personnel, and accurate, fast, and efficient express delivery ensures that the products are delivered to you quickly.