| Description | IntroductionHexokinase (HK, EC 2.7.1.1) is widely present in animals, plants, microorganisms, and cultured cells. It is the first key enzyme in the glucose degradation pathway, catalyzing the conversion of glucose to glucose-6-phosphate, which serves as the intersection point of glycolysis and the IntroductionHexokinase (HK, EC 2.7.1.1) is widely present in animals, plants, microorganisms, and cultured cells. It is the first key enzyme in the glucose degradation pathway, catalyzing the conversion of glucose to glucose-6-phosphate, which serves as the intersection point of glycolysis and the pentose phosphate pathway.Assay PrincipleHK catalyzes the synthesis of Glucose-6-Phosphate (G6P) from Glucose. Glucose-6-Phosphate Dehydrogenase (G6PDH) then further catalyzes the dehydrogenation of G6P, generating NADPH. NADPH has a characteristic absorption peak at 340 nm.Component48T96TStorageExtraction Buffer60 mL60 mL×22-8℃Reagent Ⅰ14 mL28 mL2-8℃Reagent Ⅱ1EA1EA-20℃. Store in the dark.Reagent Ⅲ1EA1EA-20℃. Store in the dark.Note: Please check the quantity of all components before starting the experiment. An additional 10% of each component is provided for standard curve preparation or pilot experiments.Required Materials and Equipment (Not Provided)TypeNameNotesInstrumentMicroplate ReaderMust be capable of measuring absorbance at 340 nmConsumables96-well UV PlateUV-transparent plateReagentsPhysiological SalineFor sample washingOtherHomogenizer (for tissue samples), Incubator, Ice box, Refrigerated centrifuge, Adjustable pipettes and tipsUsing a multichannel pipette is recommended for high-throughput experiments to improve efficiencyInstructions for Use1. Reagent Preparation试剂名称 Reagent NamePreparationNotesExtraction BufferReady-to-use; equilibrate to room temperature (RT) before use.Store at 4°CReagent ⅠReady-to-use; equilibrate to room temperature (RT) before use.Store at 4°CWorking Reagent ⅡPrepare immediately before use:• For 48T: Dissolve contents of Reagent Ⅱ in 10.8 mL Reagent Ⅰ• For 96T: Dissolve contents of Reagent Ⅱ in 21.6 mL Reagent ⅠKeep protected from light on ice during the experiment. Aliquot and store unused portions at -20°C protected from light for up to one month. Avoid repeated freeze-thaw cycles.Working Reagent ⅢPrepare immediately before use:1. Dissolve Reagent Ⅲ: • For 48T: in 0.5 mL Reagent Ⅰ • For 96T: in 1 mL Reagent Ⅰ2. Dilute the dissolved Reagent Ⅲ 10-fold with Reagent Ⅰ to make the Working Reagent Ⅲ.Keep protected from light on ice during the experiment. Aliquot and store unused portions at -20°C protected from light for up to one month. Avoid repeated freeze-thaw cycles.2. Sample PreparationNote: The use of fresh samples is highly recommended. HK activity decreases significantly upon sample freezing.2.1 Animal/Plant TissuesWeigh approximately 0.1 g of tissue. Add 1 mL of Extraction Buffer and homogenize on ice. Centrifuge the homogenate at 8000 g, 4°C for 10 min. Collect the supernatant and keep it on ice for assay.2.2 Cells, Bacteria, or FungiCollect 5×10⁶ cells/bacteria/fungi. Wash with pre-cooled physiological saline and centrifuge at 800 g for 2 min. Discard the supernatant. Add 1 mL of Extraction Buffer and disrupt the cells by sonication on ice (5 min total, 20% power or 200 W, pulse 3s on/7s off, repeat 30 times). Centrifuge the lysate at 8000 g, 4°C for 10 min. Collect the supernatant and keep it on ice for assay.2.3 Serum (or Plasma)Assay directly.3. Assay Procedure3.1 Microplate Reader Preparation: Preheat for at least 30 min. Set the wavelength to 340 nm.3.2 Working Solution Preparation: Prepare immediately before use. Each well requires 190 µL of Working Solution. It is recommended to prepare enough for 2 extra wells to account for pipetting loss.For a single well: Mix 180 µL of Working Reagent Ⅱ with 10 µL of Working Reagent Ⅲ.The Working Solution must be prepared fresh. Incubate it at 37°C for 10 min before the assay.3.3 Assay Setup: Pipette into wells of the 96-well UV plate as follows:ReagentTest Well (µL)Sample10Working Solution1903.4 Absorbance Measurement: Immediately after adding the Working Solution, mix thoroughly and measure the absorbance at 340 nm at 10 seconds (A₁) and again after exactly 10 minutes of incubation at 37°C (at 10 min 10 sec, A₂).4. Result CalculationThe following derived and simplified calculation formulas are provided and are equivalent.4.1 Data ProcessingCalculate ΔA = A₂ - A₁.4.2 Sample HK Activity Calculation① Based on Sample Mass (U/g)Definition: One unit of activity is defined as the amount of enzyme that generates 1 nmol of NADPH per minute per gram of sample.Calculation:HK (U/g) = [ΔA × Vₜₒₜₐₗ ÷ (ε × d) × 10⁹] ÷ (Vₛₐₘₚₗₑ / Vₛₐₘₚₗₑₜₒₜₐₗ × W) ÷ T × nSimplified Formula: HK (U/g) = 643.09 × ΔA ÷ W × n② Based on Cell/Bacteria/Fungi Count (U/10⁴)Definition: One unit of activity is defined as the amount of enzyme that generates 1 nmol of NADPH per minute per 10⁴ cells/bacteria/fungi.Calculation:HK (U/10⁴) = [ΔA × Vₜₒₜₐₗ ÷ (ε × d) × 10⁹] ÷ (Vₛₐₘₚₗₑ / Vₛₐₘₚₗₑₜₒₜₐₗ × 500) ÷ T × nSimplified Formula: HK (U/10⁴) = 643.09 × ΔA ÷ 500 × n③ Based on Liquid Volume (U/mL)Definition: One unit of activity is defined as the amount of enzyme that generates 1 nmol of NADPH per minute per milliliter of liquid.Calculation:HK (U/mL) = [ΔA × Vₜₒₜₐₗ ÷ (ε × d) × 10⁹] ÷ (Vₛₐₘₚₗₑ / Vₛₐₘₚₗₑₜₒₜₐₗ) ÷ T × nSimplified Formula: HK (U/mL) = 643.09 × ΔA × n④ Based on Protein Concentration (U/mg prot)Definition: One unit of activity is defined as the amount of enzyme that generates 1 nmol of NADPH per minute per milligram of protein.Calculation:HK (U/mg prot) = [ΔA × Vₜₒₜₐₗ ÷ (ε × d) × 10⁹] ÷ (Vₛₐₘₚₗₑ × Cpr) ÷ T × nSimplified Formula: HK (U/mg prot) = 643.09 × ΔA ÷ Cpr × nParameter Description:ε: NADPH molar extinction coefficient = 6.22 × 10³ L/mol/cmd: Light path of the 96-well UV plate = 0.5 cm10⁹: Conversion factor (1 mol = 1 × 10⁹ nmol)Vₜₒₜₐₗ: Total reaction volume = 200 µL = 2 × 10⁻⁴ LVₛₐₘₚₗₑ: Volume of supernatant added to the reaction = 10 µL = 1 × 10⁻⁵ LVₛₐₘₚₗₑₜₒₜₐₗ: Volume of Extraction Buffer added = 1 mLCpr: Sample protein concentration (mg/mL)W: Sample mass (g)T: Reaction time = 10 min500: Cell/Bacteria/Fungi count = 5 × 10⁶, expressed in units of 10⁴n: Sample dilution factorPrecautionsBefore formal testing, it is recommended to perform a pilot experiment using 2-3 samples expected to have significant activity differences.For tissue and cell samples, protein concentration measurement can be used to normalize results between samples.This kit is compatible with spectrophotometer detection. Adjust the preparation volumes of detection reagents proportionally according to the spectrophotometer's requirements.Biochemical reagents are generally irritating and biologically toxic. For your safety and health, please implement appropriate biosafety precautions throughout the experiment. Wear a lab coat, mask, gloves, head cover, and other protective equipment. Perform experiments in a fume hood or biosafety cabinet.This product is for scientific research use only. It is not intended for clinical diagnosis.Frequently Asked Questions (FAQ)1. What should I do if the measured ΔA is too high or too low?If ΔA > 0.5, the sample HK activity is too high. Dilute the supernatant appropriately with Extraction Buffer (include the dilution factor *n* in the calculation) or shorten the reaction time to 5 min.If ΔA < 0.005, the sample HK activity is too low. Increase the sample volume (keep the Working Solution volume constant and adjust the variable in the formula accordingly) or extend the reaction time (e.g., to 15 or 30 min).2. Can multiple samples be assayed simultaneously for high-throughput detection?The initial reaction rate is fast. It is not recommended to run a large number of samples simultaneously. If a multichannel pipette is unavailable, it is best for two individuals to perform the experiment together—one person timing and the other measuring the absorbance—to ensure the accuracy of the results... Read More | DescriptionCobalt is a transition metal that serves as a trace dietary mineral for all multicellular organisms. Cobalt is an important cofactor for the Vitamin B12class of compounds where it occupies the center of the vitamin B12corrin ring. Cobalt can also be coordinated in the active site of the DescriptionCobalt is a transition metal that serves as a trace dietary mineral for all multicellular organisms. Cobalt is an important cofactor for the Vitamin B12class of compounds where it occupies the center of the vitamin B12corrin ring. Cobalt can also be coordinated in the active site of the non-corrin containing metalloenzyme methionine aminopeptidase.Suitability: Suitable for quantitating cobalt concentrations in a variety of samplesPrinciple: The Cobalt Assay kit provides a simple and direct procedure for measuring cobalt in a variety of samples. In this assay, cobalt reacts with 2-mercaptoethanol under basic conditions to form a complex with a strong absorbance at 475 nm. Interference from the metal ions Fe2+, Cu2+, Ni2+, Zn2+, and Mn2+is <10% at this wavelength. This assay gives a linear range of 10-50 nmoles of cobalt.}Preparation instructionsSuitable for quantitating cobalt concentrations in a variety of samplesPrincipleThe Cobalt Assay kit provides a simple and direct procedure for measuring cobalt in a variety of samples. In this assay, cobalt reacts with 2-mercaptoethanol under basic conditions to form a complex with a strong absorbance at 475 nm. Interference... Read More | Inquire | DescriptioniPE-Quick Kit is intended for the advanced confirmation of target protein expression utilizingE. Coliextract before the use of theiPE kit (Prod. No. 905089) | Products contentN665730Component24 T96 TStorageN665730ATPS V50 144 µL576 µL-20℃. Avoid freeze/thaw cycle.N665730B5×FA Reaction Buffer144 µL576 µL-20℃. Avoid freeze/thaw cycle.N665730C2×HiFidelity PCR Mix600 µL2×1.2 mL-20℃. Avoid freeze/thaw Products contentN665730Component24 T96 TStorageN665730ATPS V50 144 µL576 µL-20℃. Avoid freeze/thaw cycle.N665730B5×FA Reaction Buffer144 µL576 µL-20℃. Avoid freeze/thaw cycle.N665730C2×HiFidelity PCR Mix600 µL2×1.2 mL-20℃. Avoid freeze/thaw cycle.N665730DPPM48 µL192 µL-20℃. Avoid freeze/thaw cycle.* This kit is suitable for human genomic DNA library construction with a starting template DNA input of 50 ng. We also have transposase library construction kits for human genomic DNA starting at 5 ng and 1 ng, so it is recommended to use different kits for different starting amounts of DNA in order to obtain higher quality libraries. Products IntroductionThis kit is developed for Illumina's high-throughput sequencing platform and provides the enzyme premix system and reaction buffer for genomic DNA library construction, including all components except PCR primers. Compared with the traditional library construction kits, this kit adopts the new transposase method for library construction, which can complete DNA fragmentation, end repair and junction reaction in one simple enzymatic reaction, significantly reducing the amount of template, reducing the number of experimental steps, and shortening the time of library construction; it adopts the high-fidelity DNA polymerase for library enrichment, and the preference-free PCR amplification can expand the coverage area of the sequence, which can be used for efficient and effective sequencing. The use of high-fidelity DNA polymerase for library enrichment and preference-free PCR amplification broadens the coverage area of the sequence and enables efficient preparation of DNA libraries for Illumina's second-generation sequencing platform. The kit is suitable for DNA libraries with a starting template of 50 ng, and all reagents in the kit have been subjected to strict quality control and functional validation to maximize the stability and reproducibility of library construction. Product Features ● DNA fragmentation and junction ligation in one step.● Ultra-fidelity amplification minimizes amplification-preferred steps.Provide your own instruments, kits and consumables1. Magnetic frame: DynaMagTM-2 is recommended.2. DNA purification and recovery kit: It is recommended to use DNA purification and recovery kit by magnetic bead method.3. Library PCR primer kits: transposase method for second-generation sequencing multi-sample primer kits are recommended. 4. Anhydrous ethanol, deionized water (pH between 7.0 and 8.0).5. Reaction tubes: It is recommended to use low adsorption PCR tubes and 1.5 ml centrifuge tubes. Tips: It is recommended to use high quality filter tips to prevent contamination of kits and library samples. Pre-experiment Preparation and Important Notes1. Avoid repeated freezing and thawing of reagents.2. PCR products are easily contaminated due to improper operation, resulting in inaccurate results. It is recommended to isolate the PCR reaction system preparation area from the PCR product purification area, and to use special pipettes to clean the experimental areas at regular intervals.3. Bead purification: the beads should be equilibrated to room temperature before use, all operations on the beads should be carried out at room temperature, 80% ethanol should be dispensed freshly, the beads should be rinsed and dried until the surface is free of liquid reflections and has a frosted appearance, insufficient drying of the beads will cause ethanol residue that will affect the subsequent experiments, and over-drying of the beads will affect the efficiency of DNA recovery.4. The kit is suitable for human genomic DNA library construction, if the DNA sample is a PCR product, it should be ensured that its length>.500 bp, since transposases do not work on DNA ends, it is recommended to extend the PCR product by 50-100 bp at each end of the PCR product to avoid low coverage of the ends for sequencing.Sample PreparationDNA purity requirements: A260/A280 = 1.8-2.0. Sample DNA: dissolve in ultrapure water. DNA Quantification: Too much or too little DNA will affect the quality of the library. It is recommended to use Nano to test the purity of the genomic DNA and then use Qubit to test the concentration of the genome (do not use any absorbance-based assay for template quantification).Schematic diagram of DNA banking processprocedureDNA fragmentation, junction reaction1. Add the following reagents to a 200 µl PCR tube: 2. Mix by gently blowing with a pipette and centrifuge briefly so that all components are collected at the bottom of the tube.3. Place the above PCR tubes in the PCR instrument with the hot cap on and program the reaction as follows:DNA should be purified immediately after the fragmentation reaction has been performed and the transposase is still in a high state of activity.to prevent smaller library fragments due to DNA over-fragmentation. Purification of fragmentation productsWe recommend the use of the Century Magnetic Bead Method DNA Purification and Recovery Kit.1. CMPure should be equilibrated at room temperature for 30 min after shaking and mixing before use.2. Add 50 µl of magnetic beads equilibrated to room temperature to the fragmentation product, vortex and shake for 5 seconds, then let stand at room temperature for 5 minutes.3. Centrifuge briefly, place the tube on a magnetic rack to separate the beads from the supernatant solution until the solution is clear (approximately 3-5 minutes), carefully aspirate the supernatant and discard, avoiding contact with the beads that have bound the target DNA. Note: Do not discard the beads.4. Continue to keep the centrifuge tube fixed on a magnetic rack and add 200 µl of freshly prepared 80% ethanol to the centrifuge tube and allow to stand at room temperature for 30 seconds, carefully discarding the supernatant.Note: When adding ethanol, the liquid must not be blown directly onto the beads.5. Repeat step 4.6. Keep the centrifuge tube fixed on a magnetic rack and leave to dry at room temperature until the surface of the beads is slightly cracked, then add 23 µlddH2O to solubilize.Note: Do not over-dry the beads as this may affect the elution efficiency.7. Remove the tube from the magnetic rack, vortex to completely resuspend the beads, and allow to stand at room temperature for 5 minutes. Centrifuge briefly, place the tube on the magnetic rack until the solution is clear, and transfer 21 µl of supernatant to a new 200 µl PCR tube.PCR amplification Add the following reagents to the 200 µl PCR tube: Mix by gently blowing with a pipette and centrifuge briefly so that all components are collected at the bottom of the tube.3. Place the above PCR tubes in the PCR instrument with the hot cap on and program the reaction as follows:Selective recovery of library DNA fragmentsIt is recommended to use CombiVision Magnetic Beads DNA Purification and Recovery Kit for selective recovery of DNA fragments. When different sizes of DNA fragments are required, the amount of magnetic beads to be used is different, please refer to the attached table for the specific amount of magnetic beads to be used (if other brands of magnetic beads are used, you need to find out the optimal amount of magnetic beads to be used on your own).Note: Amplification products can also be fragment length sorted and purified using the Gum Recovery Kit. If there is no special requirement for library length distribution, the amplification products can also be purified without selective recovery of DNA fragments as described on page 6 of the manual.1. CMPure should be equilibrated at room temperature for 30 min after shaking and mixing before use.2. Transfer the PCR product to a 1.5 ml centrifuge tube, rehydrate to 100 µl and add several volumes of magnetic beads equilibrated to room temperature, vortex for 5 seconds and let stand at room temperature for 5 minutes.3. Centrifuge briefly, place the tube on a magnetic rack to separate the beads from the supernatant until the solution is clear, and carefully aspirate the supernatant and transfer it to a new 1.5 ml centrifuge tube.Note: Do not discard the top clear.4. Add several volumes of magnetic beads to the supernatant, vortex and shake for 5 seconds, then let stand at room temperature for 5 minutes.5. Centrifuge briefly, place the tube on a magnetic rack to separate the beads from the supernatant until the solution is clear, carefully aspirate the supernatant and discard it, avoiding contact with the beads that have bound the target DNA.Note: Do not discard the beads.6. Continue to keep the centrifuge tube fixed on a magnetic rack and add 200 µl of freshly prepared 80% ethanol to the tube and allow to stand at room temperature for 30 seconds, carefully discarding the supernatant.Note: When adding ethanol, the liquid must not be blown directly onto the beads.7. Repeat step 6 once.8. Keep the centrifuge tube fixed on a magnetic rack and leave to dry at room temperature until the surface of the beads is slightly cracked, add 20 µl of ddH2O to solubilize.Note: Do not over-dry the beads as this may affect the elution efficiency.9. Remove the centrifuge tube from the magnetic rack, vortex and oscillate to completely resuspend the beads, and let stand at room temperature for 5 minutes. Leave brieflycentrifuge, place the tube on a magnetic rack until the solution is clear, and transfer the supernatant solution to a new centrifuge tube. Table: Suggested amount of magnetic beads for different segment selection recoveryLibrary DNA fragment purificationWe recommend the use of the Century Magnetic Bead Method DNA Purification and Recovery Kit.1. CMPure should be equilibrated at room temperature for 30 min after shaking and mixing before use.2. 50 µl of magnetic beads equilibrated to room temperature were added to the PCR product, vortexed and shaken for 5 seconds, and then left to stand at room temperature for 5 minutes.3. Centrifuge briefly, place the tube on a magnetic rack to separate the beads from the supernatant solution until the solution is clear (approximately 3-5 minutes), carefully aspirate the supernatant and discard it, avoiding contact with the beads that have bound the target DNA. Note: Do not discard the beads.4. Continue to keep the centrifuge tube fixed on a magnetic rack and add 200 µl of freshly prepared 80% ethanol to the centrifuge tube and allow to stand at room temperature for 30 seconds, carefully discarding the supernatant.Note: When adding ethanol, the liquid must not be blown directly onto the beads.5. Repeat step 4.6. Keep the centrifuge tube fixed on a magnetic rack and leave to dry at room temperature until the surface of the beads is slightly cracked, add 25 µl of ddH2O to solubilize.Note: Do not over-dry the beads as this may affect the elution efficiency.7. Remove the tube from the magnetic rack, vortex to completely resuspend the beads, and allow to stand at room temperature for 5 minutes. Centrifuge briefly, place the tube on the magnetic rack until the solution is clear, and transfer the supernatant solution to a new tube.Library quality controlDetermination of library concentrationIn order to obtain high-quality sequencing results, accurate quantification of DNA libraries is required, and the first recommendation is to use Real-timePCR methods are used for absolute quantification of DNA libraries. Additionally, fluorescent dye methods such as the Qubit method or the fluorescent dye picogreen method can be used; do not use quantification methods based on absorbance measurements here. The following approximate formula can be used to convert the molar concentration of the DNA library. Average total length of librariesApproximate conversion formula Library fragment distributionThe prepared DNA libraries can be detected by agarose gel electrophoresis or Agilent 2100 Bioanalyzer.Range of segment length distributions... Read More |