Product description
Fructose-1,6-bisphosphatase, also known as fructose 1,6-bisphosphate aldolase (FBP, EC 3.1.3.11), is a key enzyme in the gluconeogenesis pathway. Different gluconeogenic substrates are converted to 1,6-bisphosphate fructose under the action of various enzymes and then hydrolyzed to fructose 6-phosphate and inorganic phosphate by FBP. The abnormal expression of this enzyme is closely related to certain diseases. This assay kit provides a simple, sensitive, and rapid method for determination: FBP catalyzes the reaction of 1,6-bisphosphate fructose and water to produce fructose 6-phosphate and inorganic phosphate, which interacts with an enzyme complex. The NADPH produced in this process then reacts with a specific color-developing probe to form a colored substance. By detecting the increase rate of this colored substance, the activity of FBP can be calculated.
Additional Materials and Equipments Required
Microplate reader, 96-well plate, temperature-controlled water bath, desktop centrifuge, pipette, mortar and pestle, and distilled water.
FAQ
1. Transportation of Reagent Kits
Biochemical reagent kits are generally transported with ice packs. The melting of ice packs during transportation will not affect their quality, so please use them with confidence.
2. Storage of Reagent Kits
After receiving, please confirm the quantity of each component with the instructions before conducting a pre-experiment. If you have any doubts, please contact us. After opening the packaging, please store each reagent according to its specified temperature.
3. Expiry Date of Reagent Kits
The expiry date of the reagent kit is displayed on the large label, which is generally 3 months or 6 months (please refer to the large label for the exact date).
4. Pre-experiment Method
We strongly recommend that customers select 2-3 samples with significantly different expected results for pre-testing.
Purpose: To understand the situation of this batch of samples, become familiar with the experimental process, and avoid waste of experimental samples and reagents!
Step 1: Read through the paper instructions in the reagent kit, check the quantity and state (powder, liquid) of the reagents, and whether the amount of each component is consistent with the instructions. If there are no abnormalities, store them at the specified temperature.
Step 2: Take out the reagents before use, dissolve or restore to room temperature, and wait for use.
Step 3: Select 2-3 samples with significant differences for grinding and extraction, and keep the supernatant for later use.
Step 4: Conduct a pre-experiment according to the operating steps in the instructions.
Step 5: After the pre-experiment determines the sample detection concentration and adjustment, proceed with the formal experiment in batches.
5. Difference between Spectrophotometry (S) and Microplate Method (M)
S: Refers to spectrophotometry; M: Refers to microplate-based method;
Spectrophotometry refers to the use of UV spectrophotometers or visible spectrophotometers for detection. Spectrophotometry reagent kits require the use of quartz or glass cuvettes; their specifications are: light path: 1 cm, volume: 1 mL, slit width: 3 mm.
Microplate-based method refers to the use of full-wavelength continuous microplate readers for detection. Microplate method reagent kits require the use of 96-well microplates or UV plates, with specifications of: U-bottom or flat-bottom, maximum well capacity 300 μL.
6. Difference between Ordinary Microplates and UV Plates
When using a microplate reader for detection, if the wavelength is greater than or equal to 340 nm, use an ordinary 96-well plate; however, when the wavelength is less than 340 nm, a UV plate (ultraviolet microplate) is required. UV plates (PS polystyrene quartz bottom) are relatively expensive, but they can be cleaned and reused. It is generally recommended to reuse them 3-5 times.
When the final reaction solution is an organic reagent (such as ethyl acetate, etrahydrofuran, etc.), avoid using polystyrene 96-well plates.
7. Can Spectrophotometry and Microplate-based Method Reagent Kits Be Mixed?
No. Although the principles are almost the same, the reaction system, sample amount, reagent concentration, instruments, and consumables are different, and the accuracy of the results cannot be guaranteed after mixing, so it is not recommended to mix or arbitrarily change the system.
8. Is It Necessary to Remake the Standard Curve?
Generally, it is not recommended to remake the standard curve. The preparation of the standard curve will consume reagents, which will not be enough to test the number of samples specified by the reagent kit. In addition, our standard curve can be traced back, ensuring the objectivity, authenticity, and scientific rigor of the standard curve, and it can be used directly.
a. Profacgen provides the standard curve equation and the standard curve graph for indicators that require a standard curve, to prove that our standard curve is objectively made, not theoretically derived.
b. Profacgen also provides the experimental steps for drawing the standard curve. If customers have doubts about the standard curve, they can remake the standard curve according to our steps.
c. Profacgen has derived the final calculation formula based on the standard curve. Customers only need to measure the difference in absorbance and the sample weight to substitute into the calculation.
9. What Is Ice Bath Homogenization?
(1) Homogenization using a mortar: Place the mortar on an ice box for homogenization, which is ice bath homogenization. Weigh the sample and place it in the mortar, the mortar and the extraction liquid should be pre-cooled in advance or the mortar should be placed on crushed ice, an ice box, add half of the extraction liquid and grind evenly, transfer to a centrifuge tube, then add the other half of the extraction liquid to the mortar to wash the mortar, and transfer to the same centrifuge tube for merging.
(2) Homogenization using a homogenizer: First, put the grinding module in advance at -20℃ for pre-cooling; then put the weighed tissue into the centrifuge tube, add an appropriate amount of pre-cooled extraction liquid, steel beads, set the parameters according to the homogenizer requirements, and grind. Both animal and plant tissues can be used, and for samples with high fiber content and difficult to grind, the frequency and time can be increased. Or grind repeatedly multiple times.
(3) Homogenization after liquid nitrogen grinding: After the sample is ground into powder with liquid nitrogen, add the extraction liquid and grind again according to (1), (2) methods, or put it into an ultrasonic cleaner for 300W ultrasonic 5min, the temperature during the whole ultrasonic process must not exceed 10℃.
10. Sample Storage
For routine indicators, samples stored at -80℃ can be tested for over a year; stored at -40℃, they can be tested within half a year; stored at -20℃, they are fine for up to three months.