| Description | Pyruvate phosphate dikinase (PPDK, EC 2.7.9.1) is a rate-limiting enzyme in the C4 pathway and Crassulacean acid metabolism (CAM) pathway. It catalyzes the three-step conversion of ATP, pyruvate, and Pi to phosphoenolpyruvate. This enzyme is primarily located in the chloroplast stroma of C4 plants Pyruvate phosphate dikinase (PPDK, EC 2.7.9.1) is a rate-limiting enzyme in the C4 pathway and Crassulacean acid metabolism (CAM) pathway. It catalyzes the three-step conversion of ATP, pyruvate, and Pi to phosphoenolpyruvate. This enzyme is primarily located in the chloroplast stroma of C4 plants and plays a crucial regulatory role in photosynthetic function.Assay Principle The reverse reaction catalyzed by PPDK converts phosphoenolpyruvate, AMP, and PPi into pyruvate, ATP, and Pi. Lactate dehydrogenase then further catalyzes the reaction of pyruvate and NADH to produce lactate and NAD+. The decrease in NADH is measured at 340 nm, and the rate of this decrease is used to calculate PPDK activity.Component50TStorageExtraction Buffer60 mL2-8℃Reagent 160 mL2-8℃Reagent 22 EA-20℃Reagent 360 µL2-8℃Note for Reagent 3: The volume is small. If the liquid is on the tube wall, briefly centrifuge before use.User-Prepared Instruments & MaterialsUV spectrophotometer, benchtop centrifuge, adjustable pipettes, 1 ml quartz cuvette, mortar, ice, and distilled water.Sample PreparationHomogenize the tissue sample in ice-cold Extraction Buffer using a mortar and pestle, using a ratio of 1:5 to 1:10 (tissue weight (g) : Extraction Buffer volume (mL)). (It is recommended to weigh about 0.1 g of tissue and add 1 mL of Extraction Buffer). Centrifuge the homogenate at 8000 g, 4°C for 10 minutes. Collect the supernatant and keep it on ice for assay.Assay Procedure1. Spectrophotometer Setup: Preheat the spectrophotometer for at least 30 minutes. Set the wavelength to 340 nm. Zero the instrument with distilled water.2. Sample Measurement:2.1 Working Solution Preparation: Just before use, add one vial of Reagent 2 to 25 mL of Reagent 1 and add 12.5 µL of Reagent 3. Mix thoroughly and incubate at 37°C for 5 minutes. Any unused solution should be aliquoted and stored at -20°C. Avoid repeated freeze-thaw cycles.2.2 Reaction Setup: Add 50 µL of the sample supernatant and 950 µL of the Working Solution into a 1 mL quartz cuvette. Mix immediately and record the initial absorbance value at 340 nm (A1). After incubating at 37°C for exactly 5 minutes, record the absorbance value again (A2). Calculate ΔA = A1 - A2. PPDK Activity Calculation 1. Based on Sample Protein Concentration: Unit Definition: One unit of enzyme activity is defined as the amount that consumes 1 nmol of NADH per minute per mg of protein. Formula: PPDK Activity (nmol/min/mg prot) = [ΔA × V total_reaction ÷ (ε × d) × 10⁹] ÷ (V sample × Cpr) ÷ T = 643 × ΔA ÷ Cpr 2. Based on Sample Fresh Weight: Unit Definition: One unit of enzyme activity is defined as the amount that consumes 1 nmol of NADH per minute per gram of fresh tissue. Formula: PPDK Activity (nmol/min/g fresh weight) = [ΔA × V total_reaction ÷ (ε × d) × 10⁹] ÷ (W × V sample ÷ V total_extract ) ÷ T = 643 × ΔA ÷ WParameters Explanation: V total reaction : Total reaction volume, 1 × 10⁻³ L ε: Molar extinction coefficient of NADH, 6.22 × 10³ L/mol/cm d: Light path of the cuvette, 1 cm V sample : Volume of sample supernatant added, 0.05 mL V total extract : Total volume of extraction buffer added, 1 mL T: Reaction time, 5 min Cpr: Sample protein concentration, mg/mL W: Sample mass, g Notes It is essential to perform a preliminary assay using 2-3 samples expected to have significant activity differences before formal testing... Read More | Description：Acetylcholinesterases (AChEs) are enzymes that hydrolyze the neurotransmitter acetylcholine (ACh) to acetate and choline. AChE is located at the synaptic cleft and functions to terminate synaptic transmission by catalyzing the breakdown of ACh allowing cholinergic neurons to Description：Acetylcholinesterases (AChEs) are enzymes that hydrolyze the neurotransmitter acetylcholine (ACh) to acetate and choline. AChE is located at the synaptic cleft and functions to terminate synaptic transmission by catalyzing the breakdown of ACh allowing cholinergic neurons to return to a resting state after activation. It is also found in membranes of red blood cells, motor and sensory fibers, muscles, nerves and central and peripheral tissues. Changes in AChE activity may result from exposure to certain insecticides, which act as cholinesterase inhibitors. Inhibitors of AChE are also used to treat certain conditions such as dementia.Acetylcholinesterase activity assay kit has been used to determine the activity of acetylcholinesterase in a rat organophosphate model and in brain tissue homogenates.Principle：Acetylcholinesterase can catalyze the hydrolysis of acetylcholine to choline, and the reaction of choline with disulfide p-nitrobenzoic acid to produce 5-merhydryl-nitrobenzoic acid (TNB). The product has a characteristic absorption peak at 412 nm, and the activity of acetylcholinesterase can be characterized by the change of light absorption valueThe Dilution Calculator EquationConcentration (start)xVolume (start)= Concentration (final)× Volume (final)This equation is commonly abbreviated as: C1V1 = C2V2... Read More | Inquire | Store at -20°C. Please refer to protocols | Ketone bodies, 3-hydroxybutyric acid (BOH) and acetoacetic acid (AcAc), are produced in the liver primarily from oxidation of fatty acids, and are normally present at low concentrations in urine and blood. Increased ketone concentrations in the blood may lead to metabolic acidosis, which has been Ketone bodies, 3-hydroxybutyric acid (BOH) and acetoacetic acid (AcAc), are produced in the liver primarily from oxidation of fatty acids, and are normally present at low concentrations in urine and blood. Increased ketone concentrations in the blood may lead to metabolic acidosis, which has been associated with diabetes, childhood hypoglycemia, growth hormone deficiency, alcohol or salicylate intoxication, and inborn errors of metabolism.Ketone Body Assay has been used to measure the release of ketone bodies in the human liver cancer cell line HepG2 culture medium... Read More |