| Description | Pyruvate Kinase (PK, EC 2.7.1.40) is widely present in animals, plants, microorganisms, and cultured cells. It catalyzes the final step of glycolysis and is one of the key rate-limiting enzymes in glycolysis, as well as a crucial enzyme for ATP production. Therefore, determining PK activity is of Pyruvate Kinase (PK, EC 2.7.1.40) is widely present in animals, plants, microorganisms, and cultured cells. It catalyzes the final step of glycolysis and is one of the key rate-limiting enzymes in glycolysis, as well as a crucial enzyme for ATP production. Therefore, determining PK activity is of significant importance.Detection Principle: PK catalyzes the conversion of phosphoenolpyruvate and ADP to ATP and pyruvate. Lactate dehydrogenase (LDH) further catalyzes the reaction of NADH and pyruvate to produce lactate and NAD⁺. The change in NADH absorbance at 340 nm is measured to calculate the PK activity in the sample.Applicable Samples: Animal/plant tissues, cells, bacteria, serum (plasma)A1501205Component48T96TStorageA1501205AExtraction Buffer60 mL60 mL×22-8℃A1501205BAssay Buffer12 mL24 mL2-8℃A1501205CSubstrate Mix1EA1EA-20℃. Store in the dark.A1501205DLDH10.2 µL20.4 µL2-8℃Please check the quantity of each component before the experiment.An additional 10% of each component is provided beyond the specified volume for standard curve preparation or preliminary experiments.User-Provided Instruments and ReagentsTypeNameNotesInstrumentMicroplate ReaderCapable of measuring absorbance at 340 nm.Consumables96-well UV PlateUV-transparent plate.ReagentsPBS (pH 7.4) / Deionized WaterFor washing cells/bacteria / Reagent preparation.OthersHomogenizer (for tissue samples), incubator, ice bucket, low-temperature centrifuge, adjustable pipettes and tipsUsing a multichannel pipette for large-scale detection can improve efficiency.Experimental Procedure1. Reagent PreparationReagent NameReagent PreparationPrecautionsExtraction BufferReady-to-use; equilibrate to room temperature before use.Store at 4°C.Assay BufferReady-to-use; equilibrate to room temperature before use.Store at 4°C.Substrate Mix Working ReagentPrepare before use: For 48T: Add 10.2 mL Assay Buffer and 0.6 mL deionized water to the vial. For 96T: Add 20.4 mL Assay Buffer and 1.2 mL deionized water to the vial. Dissolve thoroughly.After preparation, aliquot and store at -20°C for up to 6 months. Avoid repeated freeze-thaw cycles. Before use, incubate at 25°C (for general species) or 37°C (for mammals) for 5 min.LDH Working ReagentPrepare before use: For 48T: Add 0.6 mL deionized water to the LDH vial. For 96T: Add 1.2 mL deionized water to the LDH vial. Mix thoroughly.Keep on ice after preparation. The diluted reagent can be stored at 4°C for 1 month.2. Sample PreparationNote: Fresh samples are recommended. If not used immediately, samples can be stored at -80°C for up to 1 month.2.1 Animal/Plant Tissues: Weigh approximately 0.1 g of tissue, add 1 mL of Extraction Buffer, and homogenize on ice. Centrifuge at 8000 g, 4°C for 10 minutes. Collect the supernatant and keep on ice for detection.2.2 Cells/Bacteria: Collect 5×10⁶ cells or bacteria into a centrifuge tube. Wash with cold PBS, centrifuge, and discard the supernatant. Add 1 mL of Extraction Buffer. Disrupt by ultrasonic homogenization on ice (power 20% or 200 W, ultrasonicate for 3 s, interval 7 s, repeat 30 times). Centrifuge at 8000 g, 4°C for 10 minutes. Collect the supernatant and keep on ice for detection.2.3 Serum (Plasma) and other liquid samples: Detect directly.3. Assay Steps3.1 Microplate Reader Preparation: Preheat for at least 30 minutes. Set the wavelength to 340 nm.3.2 Assay System Setup: In a 96-well UV plate, add sequentially: 10 µL of sample, 10 µL of LDH Working Reagent, and 180 µL of Substrate Mix Working Reagent. Mix rapidly immediately after addition.3.3 Absorbance Measurement: Immediately after mixing, measure the absorbance at 340 nm at 20 seconds (A1) and then at 2 minutes and 20 seconds (A2). Calculate ΔA = A1 - A2.4. Result Calculation4.1 Data ProcessingCalculate ΔA = A1 - A2.4.2 Sample PK Activity Calculation(1) Based on sample mass:Unit Definition: One unit of enzyme activity is defined as the consumption of 1 nmol NADH per minute per gram of tissue in the reaction system.Formula:PK (U/g) = [ΔA×Vtotal reaction÷ (ε × d) × 10⁹] ÷ (Vsample ÷ Vtotal extract×W) ÷ T = 3215 × ΔA ÷ W(2) Based on cell/bacterial count:Unit Definition: One unit of enzyme activity is defined as the consumption of 1 nmol NADH per minute per 10⁴ bacteria or cells in the reaction system.Formula:PK (U/10⁴) = [ΔA × Vtotal reaction ÷ (ε × d) × 10⁹] ÷ (Vsample ÷ Vtotal extract; × 500) ÷ T = 3215 × ΔA ÷ 500 = 6.431 × ΔA(3) Based on liquid volume:Unit Definition: One unit of enzyme activity is defined as the consumption of 1 nmol NADH per minute per mL of serum (plasma) in the reaction system.Formula:PK (U/mL) = [ΔA × Vtotal reaction; ÷ (ε × d) × 10⁹] ÷ Vsample ÷ T = 3215 × ΔA(4) Based on protein concentration:Unit Definition: One unit of enzyme activity is defined as the consumption of 1 nmol NADH per minute per mg of protein in the reaction system.Formula:PK (U/mg prot) = [ΔA × Vtotal reaction; ÷ (ε × d) × 10⁹] ÷ (Cpr × Vsample ) ÷ T = 3215 × ΔA ÷ CprParameter Description:Vtotal reaction; : Total reaction volume, 2 × 10⁻⁴ Lε: Molar extinction coefficient of NADH, 6.22 × 10³ L/mol/cmd: Light path of the 96-well plate, 0.5 cm10⁹: Conversion factor (1 mol = 1 × 10⁹ nmol)Vsample : Volume of sample added, 0.01 mLVtotal extract; : Volume of Extraction Buffer added, 1 mLT: Reaction time, 2 minCpr: Sample protein concentration, mg/mLW: Sample mass, g500: Cell or bacterial count (5 × 10⁶), converted to units of 1Precautions1. It is recommended to perform preliminary experiments using 2-3 samples expected to have significant differences before formal testing.2. This kit is compatible with spectrophotometer detection. Adjust the preparation volume of detection reagents proportionally according to the spectrophotometer's requirements.3. For tissue and cell samples, results can be normalized by measuring the protein concentration. Aladdin BCA Protein Quantification Kit (B665595) or Ready-to-Use BCA Protein Quantification Kit (R1491648) are recommended.4. Biochemical reagents are generally irritating and biologically toxic. For your safety and health, please implement appropriate biosafety precautions throughout the experiment. Wear personal protective equipment such as lab coats, masks, gloves, and hair caps. Perform experiments in a fume hood or biosafety cabinet.5. This product is for scientific research use only. Not intended for clinical diagnosis.Frequently Asked QuestionsQ: What should I do if the sample ΔA is too high or too low?A: If the sample ΔA is > 1.0, the PK activity in the sample is too high. Dilute the sample appropriately with Extraction Buffer or reduce the amount of sample used for extraction, and then re-assay. If the sample ΔA is < 0.005, extend the reaction time to 5 or 10 minutes, or appropriately increase the sample amount, and then re-assay.Q: Will testing multiple samples simultaneously affect the results?A: Appropriately extending the time by 3-5 minutes for this assay will not affect the results. When testing multiple samples, it is recommended to use a multi-channel pipette for operation... Read More | Alanine Aminotransferase (ALT), also known as serum glutamic-pyruvic transaminase (SGPT), is a pyridoxal-phosphate-dependent enzyme that catalyzes the reversible transfer of an amino group from alanine to α-ketoglutarate, generating pyruvate and glutamate. ALT is found primarily in liver and Alanine Aminotransferase (ALT), also known as serum glutamic-pyruvic transaminase (SGPT), is a pyridoxal-phosphate-dependent enzyme that catalyzes the reversible transfer of an amino group from alanine to α-ketoglutarate, generating pyruvate and glutamate. ALT is found primarily in liver and serum, but occurs in other tissues as well. Hepatocellular injury often results in an increase of serum ALT levels and serum ALT levels can be used as a marker for liver injury.ALT Activity Assay kit has been used to determine the activity of alanine aminotransferase (ALT) in serum samples... Read More | Inquire | DescriptionUse in combination with the KitAlysis Bench Top Inertion Box (Z742064) or a glove box/glove bag to provide inert atmosphere for kit set-up.Designed to be used with KitAlysis High-Throughput Screening Kits.Components:24-Well Reaction BlockTorque ScrewdriverSmall screwdriver to easily DescriptionUse in combination with the KitAlysis Bench Top Inertion Box (Z742064) or a glove box/glove bag to provide inert atmosphere for kit set-up.Designed to be used with KitAlysis High-Throughput Screening Kits.Components:24-Well Reaction BlockTorque ScrewdriverSmall screwdriver to easily remove torqued screws after reaction is complete.10 Reaction Block Replacement Screws... Read More | Product introduction：Reporter gene detection is an important tool for analyzing the interaction between potential cis elements (such as promoters, enhancers and silencers) and trans acting factors in the flanking region of structural genes in the field of modern molecular biology. Firefly Product introduction：Reporter gene detection is an important tool for analyzing the interaction between potential cis elements (such as promoters, enhancers and silencers) and trans acting factors in the flanking region of structural genes in the field of modern molecular biology. Firefly luciferase is widely used in gene regulation and drug screening. Firefly luciferase is a protein with a molecular weight of about 61 KD. In the presence of ATP, magnesium ions and oxygen, it can catalyze the production of oxyluciferin from luciferin. In the process of luciferin oxidation, it will produce a light signal. The optical signal of this kit is a kind of instantaneous light, which needs to be detected immediately after adding the working solution. The half-life of optical signal is about 5 min.Instruction：1.Working fluid configuration ( 1 ) Restore all components to room temperature. ( 2 ) The component B ( stock solution ) was fully diluted with component A to prepare a 0.2 mg / mL firefly luciferase working solution, which was vortexed and shaken to ensure full mixing. Note : The firefly luciferase working solution cannot be repeatedly frozen and thawed. If the dosage of a single experiment is small, it is recommended to subpackage according to a single dosage. At room temperature, the activity decreased by about 10 % after the working solution was configured for 3 h, and the activity decreased by about 25 % after 5 h. 2.chemiluminescence value detection ( 1 ) The cell culture plate was taken out from the incubator and incubated at room temperature for 20 min to restore it to room temperature ( 22-25 ° C ). ( 2 ) Add the same volume of firefly luciferase working solution with the medium to the culture plate and mix well. ( 3 ) Incubation at room temperature for 5 min. Note : The incubation time can be adjusted according to cell type and cell number. ( 4 ) The values were read by multifunctional microplate reader or chemiluminescence instrument ( instrument parameters : the determination time was 10 s, the determination interval was 2 s ).Matters needing attention：1. please centrifuge the product to the bottom of the tube immediately before use, and then conduct subsequent experiments. 2. the strongest wavelength of bioluminescence catalyzed by firefly luciferase is 560 nm. 3. to prevent interference between holes, it is recommended to use white opaque orifice plate.Recommendation：Component B is recommended to use sterile water in advance to configure 2 mg / mL storage solution, A component and B component configured as storage solution, and small batch packaging according to the experimental requirements. The detection working fluid is recommended to be used now to avoid repeated freezing and thawing. Component：One-Step Firefly Luciferase Assay Buffer；D-Luciferin Scope of application：Mainly used for ADCC detection... Read More |