| Description | Acetate Kinase (ACK) is primarily found in microorganisms. It catalyzes the conversion of acetate and ATP to acetyl phosphate and ADP, serving as a key enzyme in bacterial carbon and energy metabolism, and plays a central role particularly in the methanogenesis metabolism of archaea.Assay Acetate Kinase (ACK) is primarily found in microorganisms. It catalyzes the conversion of acetate and ATP to acetyl phosphate and ADP, serving as a key enzyme in bacterial carbon and energy metabolism, and plays a central role particularly in the methanogenesis metabolism of archaea.Assay PrincipleACK catalyzes the synthesis of Acetyl Phosphate and ADP from Sodium Acetate and ATP. Pyruvate Kinase then catalyzes the conversion of ADP and Phosphoenolpyruvate (PEP) to ATP and Pyruvate. Subsequently, Lactate Dehydrogenase catalyzes the reduction of Pyruvate by NADH to produce Lactate and NAD⁺. The rate of oxidation of NADH to NAD⁺, measured by the decrease in absorbance at 340 nm, reflects ACK activity.Component48T96TStorageExtraction Buffer60 mL60 mL×22-8℃ReagentⅠ15 mL30 mL2-8℃ReagentⅡ1EA2EA-20℃. Store in the dark.Reagent III25 µL50 µLNote: It is recommended to perform a pilot experiment with 2-3 samples expected to have significant differences before formal testing.Required Materials and Equipment (Not Provided)Microplate reader or UV spectrophotometer (capable of measuring absorbance at 340 nm)96-well UV plate or micro quartz cuvetteAdjustable pipettes and tipsConstant temperature water bathIce makerCentrifugeDeionized waterHomogenizer (for tissue samples)Reagent PreparationExtraction Buffer: Ready-to-use. Equilibrate to room temperature (RT) before use. Store at 4°C.Caution: Extraction Buffer is toxic and has a pungent odor. It is recommended to handle it within a fume hood.Reagent Ⅰ: Ready-to-use. Equilibrate to RT before use. Store at 4°C.Working Reagent Ⅱ: Prepare immediately before use. For one vial of Reagent Ⅱ, add 11 mL of Reagent Ⅰ and 19.8 µL of Reagent III. Mix thoroughly to dissolve. Prepare fresh for each use. Can be stored protected from light at -20°C for one month.Reagent Ⅲ: Ready-to-use. Equilibrate to RT before use. Store at 4°C protected from light.Sample Preparation*Note: The use of fresh samples is recommended. If not used immediately, samples can be stored at -80°C for up to one month. Control the temperature and time during thawing. If thawed at room temperature, complete the process within 4 hours.*1.Tissues: Weigh approximately 0.1 g of sample. Add 1 mL of Extraction Buffer and homogenize on ice. Centrifuge the homogenate at 15,000 g, 4°C for 10 min. Collect the supernatant and keep it on ice for assay.2.Cells or Bacteria: Collect 5 million cells or bacteria by centrifugation. Wash the pellet with cold PBS, centrifuge, and discard the supernatant. Add 1 mL of Extraction Buffer. Disrupt the cells/bacteria by sonication on ice (200W power, pulse 3s on/10s off, repeat 30 times). Centrifuge the lysate at 15,000 g, 4°C for 10 min. Collect the supernatant and keep it on ice for assay.3.Serum (Plasma) or other liquid samples: Assay directly. If the solution is turbid, centrifuge first and use the supernatant for assay.Note: To determine protein concentration, Aladdin's BCA Protein Quantification Kit (B665595) or Ready-to-Use BCA Protein Quantification Kit (R1491648) is recommended.Assay Procedure1.Preheat the microplate reader or spectrophotometer for 30 min. Set the wavelength to 340 nm. Zero the spectrophotometer with deionized water.2.Pre-warm a sufficient volume of the prepared Working Reagent Ⅱ at 37°C (for mammalian samples) or 25°C (for other species) for 5 minutes. Use immediately.3.Assay Setup (perform in a 96-well UV plate or micro quartz cuvette):ReagentTest Well (µL)Sample20Working Reagent Ⅱ180Mix thoroughly immediately after addition. Measure the absorbance at 340 nm at 10 seconds (A₁) and again at 190 seconds (A₂). Calculate ΔA = A₁ - A₂.Note: It is advised to run a pilot test with 2-3 samples showing expected significant variation beforehand. If ΔA is less than 0.05, consider increasing the sample volume or extending the reaction time to 10 or 20 minutes before measurement. If ΔA is greater than 1.0, dilute the sample further with Extraction Buffer (multiply the result by the dilution factor) or reduce the amount of sample used for extraction.Result CalculationNote: Both the derived and simplified calculation formulas are provided and are equivalent. The simplified formulas (in bold) are recommended for final calculation.1. Calculation for 96-Well UV PlateGeneral Parameters for 96-Well Plate:ε (NADH molar extinction coefficient) = 6.22 × 10³ L/mol/cmd (Light path of 96-well plate) = 0.5 cmVₜₒₜₐₗ (Total reaction volume) = 0.0002 L (200 µL)Vₛₐₘₚₗₑ (Sample volume in reaction) = 0.02 mL (20 µL)T (Reaction time) = 3 minVₛₐₘₚₗₑₜₒₜₐₗ (Total extraction volume) = 1 mLCpr (Sample protein concentration, mg/mL)W (Sample mass, g)500 (Cell/Bacteria count in millions: 5 × 10⁶)1.1 Based on Sample Protein Concentration:Definition: One unit of activity is defined as the amount of enzyme that consumes 1 nmol of NADH per minute per mg of protein.Calculation:ACK Activity (U/mg prot) = [ΔA × Vₜₒₜₐₗ ÷ (ε × d) × 10⁹] ÷ (Vₛₐₘₚₗₑ × Cpr) ÷ TSimplified Formula: ACK (U/mg prot) = 1072 × ΔA ÷ Cpr1.2 Based on Sample Mass:Definition: One unit of activity is defined as the amount of enzyme that consumes 1 nmol of NADH per minute per gram of fresh sample.Calculation:ACK Activity (U/g fresh weight) = [ΔA × Vₜₒₜₐₗ ÷ (ε × d) × 10⁹] ÷ (W × Vₛₐₘₚₗₑ / Vₛₐₘₚₗₑₜₒₜₐₗ) ÷ TSimplified Formula: ACK (U/g fresh weight) = 1072 × ΔA ÷ W1.3 Based on Bacterial or Cell Density:Definition: One unit of activity is defined as the amount of enzyme that consumes 1 nmol of NADH per minute per 10⁴ cells/bacteria in the reaction system.Calculation (for 5 million cells in 1 ml extract):ACK Activity (U/10⁴) = [ΔA × Vₜₒₜₐₗ ÷ (ε × d) × 10⁹] ÷ (500 × Vₛₐₘₚₗₑ / Vₛₐₘₚₗₑₜₒₜₐₗ) ÷ TSimplified Formula: ACK (U/10⁴) = 2.144 × ΔA1.4 Based on Liquid Volume:Definition: One unit of activity is defined as the amount of enzyme that consumes 1 nmol of NADH per minute per milliliter of sample.Calculation:ACK Activity (U/mL) = [ΔA × Vₜₒₜₐₗ ÷ (ε × d) × 10⁹] ÷ Vₛₐₘₚₗₑ ÷ TSimplified Formula: ACK (U/mL) = 1072 × ΔA2. Calculation for Micro Quartz CuvetteUse the formulas above but adjust the light path *d* from 0.5 cm to 1.0 cm.Precautions1. Keep samples and all reagents on ice during the assay procedure to prevent denaturation and loss of activity.2. The temperature of the reaction mixture must be maintained at 37°C or 25°C. When using a cuvette, a small beaker filled with deionized water pre-warmed to 37°C or 25°C (placed in a water bath) can be used to hold the cuvette and maintain temperature during the reaction.3. This product is for scientific research use only. It is not intended for clinical diagnosis. For your safety and health, please wear a lab coat and disposable gloves during operation... Read More | Format:2-ComponentEnzyme:Horseradish peroxidase | This reagent kit is for research purposes only. Purpose of use: This reagent kit is used to determine the content of lactose in serum, plasma, and related liquid samples.Experimental principle:This kit applies enzyme-linked immunosorbent assay to determine the level of lactose in the sample. This reagent kit is for research purposes only. Purpose of use: This reagent kit is used to determine the content of lactose in serum, plasma, and related liquid samples.Experimental principle:This kit applies enzyme-linked immunosorbent assay to determine the level of lactose in the sample. Purified lactose antibodies were coated on a microplate to produce solid-phase antibodies. Lactose was added to the microplate of the coated monoclonal antibody, along with HRP labeled lactose antigens, to compete for binding. After thorough washing, the substrate TMB was added for colorimetry. The depth of sample color is negatively correlated with the content of lactose in the sample. Measure the absorbance (OD value) at a wavelength of 450nm using an enzyme-linked immunosorbent assay (ELISA) reader, and calculate the content of lactose in the sample through a standard curve.Kit composition:130times concentrated washing solution20ml×1 bottle8.1Standard S1（80µg/L）0.5ml×1bottle2Enzyme-linked immunosorbent assay6ml×1 bottle8.2Standard S2（40µg/L）0.5ml×1bottle3Enzyme labeling coated plate96 holes x 1 pieces8.3Standard S3（20µg/L）0.5ml×1bottle4Color reagent A solution6ml×1 bottle8.4Standard S4（10µg/L）0.5ml×1bottle5Color developer B solution6ml×1 bottle8.5Standard S5（5µg/L）0.5ml×1bottle6Stop solution6ml×1 bottle9Instructions1 copy7Sample Diluent6ml×1 bottle10Microplate Sealers2 sheetsSpecimen requirements:1. Specimen processing:(1) After collecting the water sample, it is repeatedly freeze-thawed three times at -20 ℃, and then filtered through glass fiber for future reference(2) The tissue samples should be extracted using butanol: methanol: water (5:25:70 V: V: V), or extracted according to relevant literature. The experiment should be conducted as soon as possible after extraction. If the experiment cannot be conducted immediately, the specimen can be stored at -20 ℃ for future reference2. Samples containing NaN3 cannot be detected as NaN3 inhibits the activity of horseradish peroxidase (HRP).Operation steps:1. Sample addition: Set up standard wells, blank wells (blank control wells do not include samples and enzyme-linked immunosorbent assay reagents, the other steps are the same), and sample wells to be tested. Add 50 microliters to the standard well on the enzyme-linked immunosorbent assay (ELISA) plate, and first add 40 diluents to the sample well to be tested µ l. Then add 10 more samples to be tested µ L (The final dilution of the sample is 5 times). Add the sample to the bottom of the enzyme-linked immunosorbent assay (ELISA) plate well, avoiding touching the well wall as much as possible. Gently shake and mix well.2. Enzyme addition: Add 50 enzyme labeled reagents to each well µ l. Excluding blank holes.3. Warm incubation: Seal the plate with a sealing film and incubate at 37 ℃ for 60 minutes.4. Solution preparation: Dilute 30 times the concentrated washing solution with distilled water and set aside for later use5. Washing: Carefully remove the sealing film, discard the liquid, shake dry, fill each well with washing solution, let it stand for 30 seconds, then discard. Repeat this process 5 times and pat dry.6. Color development: Add color development agent A50 to each well first µ l. Add color developer B50 again µ l. Gently shake and mix well, and develop color at 37 ℃ in the dark for 15 minutes7. Termination: Add 50% termination fluid to each hole µ l. Terminate the reaction (at this point, the blue color immediately turns yellow).8. Measurement: Zero the blank hole and sequentially measure the absorbance (OD value) of each hole at a wavelength of 450nm. The measurement should be conducted within 15 minutes after adding the termination solution.Calculation:Draw a standard curve on a coordinate paper with the concentration of the standard substance as the x-axis and the OD value as the y-axis. Based on the OD value of the sample, determine the corresponding concentration from the standard curve; Multiply it by the dilution factor; Alternatively, a linear regression equation can be used to calculate the standard curve using the concentration and OD value of the standard substance. The OD value of the sample can be substituted into the equation to calculate the sample concentration, which is then multiplied by the dilution factor to obtain the actual concentration of the sample.Notes:1. The kit should be balanced at room temperature for 15-30 minutes before use when taken out from the cold storage environment. If the enzyme coated plate is not used up after opening, the Flat noodles should be stored in a sealed bag.2. Concentrated detergent may precipitate crystals. When diluted, it can be heated in a water bath to aid in dissolution. Washing does not affect the results.3. A sampler should be used for each step of sample addition, and its accuracy should be regularly calibrated to avoid experimental errors. It is best to control the sample addition time within 5 minutes. If there are a large number of specimens, it is recommended to use a firing gun for sample addition.4. Please make a standard curve at the same time as each measurement, preferably with a re hole. If the content of the substance to be tested in the sample is too high (the OD value of the sample is greater than the OD value of the first well of the standard well), please dilute the sample diluent by a certain multiple (n times) before measurement. When calculating, please multiply the total dilution multiple (x n x 5).5. The sealing film is only for one-time use to avoid cross contamination.6. Please store the substrate in dark.7. Strictly follow the instructions and determine the test results based on the reading of the enzyme-linked immunosorbent assay (ELISA) reader8. All samples, washing liquids, and various waste should be treated as infectious substances.9. The components of this reagent with different batch numbers shall not be mixed.Detection range:two µ G/L-90 µ G/L... Read More | The content of this cell is too long for an XLSX file (more than 32767 characters). Please use the CSV format for this export | Products contentProducts IntroductionThe Single Cell Whole Genome Amplification Kit can be used as a template for whole genome amplification of single cells or micro samples. The total time for single-cell amplification is about 3 hours, and 2-5 µg of genomic DNA, with a size of 200-1500 bp, Products contentProducts IntroductionThe Single Cell Whole Genome Amplification Kit can be used as a template for whole genome amplification of single cells or micro samples. The total time for single-cell amplification is about 3 hours, and 2-5 µg of genomic DNA, with a size of 200-1500 bp, can be obtained after lysis, pre-amplification and amplification. The amplified product can be widely used in second-generation sequencing, large fragment copy number variation analysis, SNP typing, qPCR analysis and gene chip analysis.Bring your own instruments and reagentsPCR instrument Reaction tubes: low adsorption tubes recommended Gun Heads: High quality filtered gun heads are recommended Microcentrifuge, vortex mixercaveat The sensitivity of this product is very high, the experimental operation should be completed in a positive pressure ultra-clean bench or clean environment, the concentration of the amplification reaction products is high, should be well isolated to avoid aerosol contamination caused by amplification products.Operation flow diagramprocedurePre-experiment preparationSingle cells were obtained by flow cytometry sorting, buffer dilution, micromanipulation and laser microdissection. It is recommended that the cells be washed prior to the experiments with a 1× PBS solution free of Mg2+ and Ca2+, taking care to ensure that the volume of PBS solution in subsequent experiments does not exceed 2 µl. take note of Since the whole experiment is carried out in the same PCR tube and the reaction volume is small, the pipette tip should not touch the liquid in the tube when adding liquid, so as to avoid taking single cells or DNA out of the reaction system; when pipetting, please add the liquid along the wall of the tube carefully and do not blow the liquid in the PCR tube; before the reaction, please centrifuge briefly to make sure that the liquid in the reaction system is mixed evenly. Thaw the cell lysate, pre-amplifier and amplifier on ice before use.cell lysis 1）Mix Cell Lysis Buffer and Cell Lysis Enzyme according to the number of reactions N, shake to mix, centrifuge briefly and set aside.2）Mix single cells with the cell lysis mix in a PCR tube and run the following program.2. Pre-amplification reaction1）Mix Cell Lysis Buffer and Cell Lysis Enzyme according to the number of reactions N, shake to mix, centrifuge briefly and set aside.2）Add 5 µl of pre-amplification mix to 10 µl of lysis reaction product from the previous step and run the following program. 3. Amplification reaction1）Mix Amplification Buffer and Amp Enzyme Mix according to the number of reactions N, mix with shaking, centrifuge briefly and set aside.2）Add 60 µl of amplification mix to 15 µl of pre-amplification reaction product from the previous step and run the following program.Note: The number of cycles can be adjusted as needed, 14 cycles are recommended for single cells obtained by flow sorting, etc.Amplification product detection 1. Agarose gel electrophoresis 5 µl of the amplified product was subjected to agarose gel electrophoresis (1% agarose gel, 110 V, 25-35 min), and the amplified product was 200-1500 bp in size. 2. Quantitative Amplification products were subjected to magnetic bead or column purification, and purified products were quantified using Qubit with a final yield of 2-5 µg... Read More |