| 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.Component50TStorageExtraction Buffer80 mL2-8℃Reagent 11EA-20℃Reagent 24EA-20℃Reagent 32EA-20℃Reagent 42EA-20℃Reagent PreparationReagent 1: Before use, centrifuge the vial to bring the powder to the bottom. Add 8.4 mL of distilled water to dissolve. Aliquot and store unused portions at -20°C.Reagent 2: Before use, centrifuge each vial to bring the powder to the bottom. Add 0.6 mL of distilled water to each vial to dissolve. Aliquot and store unused portions at -20°C. Avoid repeated freeze-thaw cycles. Use within 3 days.Reagent 3: Before use, centrifuge each vial to bring the powder to the bottom. Add 0.6 mL of distilled water to each vial to dissolve thoroughly. Can be aliquoted and frozen. Avoid repeated freeze-thaw cycles.Reagent 4: Before use, centrifuge each vial to bring the powder to the bottom. Add 0.6 mL of distilled water to each vial to dissolve thoroughly. Can be aliquoted and frozen. Avoid repeated freeze-thaw cycles.Required Materials and Equipment (Not Provided)UV spectrophotometer, 1 mL quartz cuvette (1 cm light path), refrigerated benchtop centrifuge, constant temperature incubator, pipettes, mortar and pestle, ice, and distilled water.Sample Preparation1.Tissue Samples: Weigh approximately 0.1 g of tissue. Add 1 mL of Extraction Buffer and homogenize on ice. Centrifuge the homogenate at 12,000 rpm (approx. 13,000-15,000 g), 4°C for 10 min. Collect the supernatant and keep it on ice for assay.Note: For larger samples, use an Extraction Buffer volume (mL) to tissue mass (g) ratio between 5:1 and 10:1.2.Bacteria/Cell Samples: Collect cells by centrifugation and discard the supernatant. Add 1 mL of Extraction Buffer per 5 million cells/bacteria. Disrupt the cells/bacteria by sonication on ice (20% power or 200W, pulse 3s on/10s off, repeat 30 times). Centrifuge the lysate at 12,000 rpm, 4°C for 10 min. Collect the supernatant and keep it on ice for assay.Note: For larger samples, use an Extraction Buffer volume (mL) to cell count (10⁴ cells) ratio between 1:1000 and 1:5000.Assay Procedure:1.Preheat the UV spectrophotometer for at least 30 minutes. Set the wavelength to 340 nm. Zero the instrument with distilled water.2.Pre-warm all reagents at 37°C for 5-15 minutes.3.Optional Master Mix: A Master Mix can be prepared just before use by combining Extraction Buffer, Reagent 1, Reagent 2, Reagent 3, and Reagent 4 in a 400:160:40:20:20 ratio (e.g., for one assay: 400µL + 160µL + 40µL + 20µL + 20µL = 640µL). Pipette 640 µL of this Master Mix per cuvette. Prepare the Master Mix fresh for immediate use.4.Pipette into a 1 mL quartz cuvette (1 cm light path) in the following order:ReagentVolume (µL)Extraction Buffer400Reagent 1160Reagent 240Reagent 320Reagent 420Mix thoroughly and incubate at 37°C for 5 min.Sample60Mix thoroughly immediately after adding the sample. Record the initial absorbance (A₁) at 340 nm at 10 seconds. Record the absorbance again (A₂) after exactly 10 minutes. Calculate ΔA = A₁ - A₂.Notes & Troubleshooting:1.If ΔA is close to zero, consider extending the reaction time (e.g., to 20 min) to read A₂. Use the new reaction time (T) in the calculation. Alternatively, increase the sample volume V₁ (e.g., to 100 µL, decrease Extraction Buffer accordingly) and use the new V₁ and T in the calculation.2.If the initial absorbance A₁ is too high (e.g., >2, common in deeply pigmented plant samples), reduce the sample volume V₁ and use the new V₁ in the calculation. Alternatively, add a small amount of activated charcoal to the sample supernatant, mix, let stand for 5 min, centrifuge at 12,000 rpm, 4°C for 10 min, and use the clarified supernatant for assay.3.If ΔA is greater than 0.6, reduce the reaction time (e.g., to 5 min) and use the new time (T) in the calculation.4.If the decrease is not linear, read the absorbance every 10 seconds and select a linear segment for calculating ΔA. Use the corresponding time interval (T) for the calculation.ACK Activity Calculation:General Parameters:ε (NADH molar extinction coefficient) = 6.22 × 10³ L/mol/cmd (Cuvette light path) = 1 cmV (Total extraction volume) = 1 mLV₁ (Sample volume in reaction) = 0.06 mL (60 µL)V₂ (Total reaction volume) = 0.0007 L (700 µL)T (Reaction time) = 10 min500 (Cell/Bacteria count in ten-thousands: 500 × 10⁴ = 5 million)W (Sample mass, g)Cpr (Sample protein concentration, mg/mL)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 (nmol/min/mg prot) = [ΔA ÷ (ε × d) × V₂ × 10⁹] ÷ (Cpr × V₁ / V) ÷ TSimplified Formula: ACK (nmol/min/mg prot) = 187.6 × ΔA ÷ Cpr2. Based on Sample Fresh Weight:Definition: One unit of activity is defined as the amount of enzyme that consumes 1 nmol of NADH per minute per gram of fresh tissue.Calculation:ACK Activity (nmol/min/g fresh weight) = [ΔA ÷ (ε × d) × V₂ × 10⁹] ÷ (W × V₁ / V) ÷ TSimplified Formula: ACK (nmol/min/g fresh weight) = 187.6 × ΔA ÷ W3. Based on Bacterial/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.Calculation (for 5 million cells in 1 ml extract):ACK Activity (nmol/min/10⁴ cell) = [ΔA ÷ (ε × d) × V₂ × 10⁹] ÷ (500 × V₁ / V) ÷ TSimplified Formula: ACK (nmol/min/10⁴ cell) = 0.38 × ΔAPrecautionsBefore formal assay, it is essential to perform a pilot test with 2-3 samples expected to have significant differences in activity... Read More | DescriptionIt contains a set of seven different homogeneous palladium catalysts, useful for rapid screening of catalysis conditions. It is in sampler format with individual components packaged for multiple experiments and mini scale-up. The cost of the kit is less than the total cost of individual DescriptionIt contains a set of seven different homogeneous palladium catalysts, useful for rapid screening of catalysis conditions. It is in sampler format with individual components packaged for multiple experiments and mini scale-up. The cost of the kit is less than the total cost of individual components.Catalysis Screening Kits... Read More | Products contentN665737Component24 T96 TStorageN665737ATPS V50 168 µL672 µL-20℃. Avoid freeze/thaw cycle.N665737B5×FA Reaction Buffer96 µL384 µL-20℃. Avoid freeze/thaw cycle.N665737CTS Buffer72 µL288 µL-20℃. Avoid freeze/thaw cycle.N665737D2×Products contentN665737Component24 T96 TStorageN665737ATPS V50 168 µL672 µL-20℃. Avoid freeze/thaw cycle.N665737B5×FA Reaction Buffer96 µL384 µL-20℃. Avoid freeze/thaw cycle.N665737CTS Buffer72 µL288 µL-20℃. Avoid freeze/thaw cycle.N665737D2×PCR Mix600 µL2×1.2 mL-20℃. Avoid freeze/thaw cycle. * This kit is suitable for human genomic DNA library construction, the starting template DNA input is 5 ng. We also have transposase library construction kits for 50 ng and 1 ng of human genomic DNA starting, in order to get a higher quality library, it is recommended to use different kits for different starting amount of DNA. 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 use with a starting template DNA input of 5 ng, and all reagents in the kit have been subjected to stringent 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 preference.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 kit: It is recommended to use transposase method for second generation sequencing multi-sample primer kit.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 reaction 1. 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: inactivation reactionAfter the DNA is fragmented, the enzyme is still in a high active state, so it should be removed from the PCR instrument immediately and terminated by adding the Reaction Termination Buffer, in order to prevent the DNA from being fragmented too much and resulting in smaller library fragments.1. Add 3 µl of TS Buffer to the PCR tube containing the fragmentation product.2. Mix by gently blowing with a pipette and centrifuge briefly so that all components are collected at the bottom of the tube.3. Incubate at room temperature for 5 min, or if the room temperature is too low, place the reaction on a PCR instrument at 25°C with the thermal cover closed.1. 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 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 used is different, please refer to the attached table for the specific amount of magnetic beads used.(If using other brands of magnetic beads, you need to figure out the optimal amount of magnetic beads by yourself).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, amplification products can also be purified directly from DNA fragments without selective recovery of DNA fragments as described on page 4 of the manual.1. CMPure should be equilibrated at room temperature for 30 min after shaking and mixing before use.2. Transfer the PCR products to a 1.5 ml centrifuge tube, rehydrate to 100 µl, 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 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 centrifuge tube. Table: Suggested amount of magnetic beads for different segment selection recovery Library 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 ddH O 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. Library fragment distributionThe prepared DNA libraries can be detected by agarose gel electrophoresis or Agilent 2100 Bioanalyzer.Range of segment length distributions... 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 | DescriptionGreen LED Array for Photo KitAlysis high-throughput screening platform. For use with Photo KitAlysis Starter Kit (Z742612). User guide is provided in the below hyperlink.Photo KitAlysis Operating InstructionsFeatures:Designed and tested by synthetic chemists.Controller provides repeatableDescriptionGreen LED Array for Photo KitAlysis high-throughput screening platform. For use with Photo KitAlysis Starter Kit (Z742612). User guide is provided in the below hyperlink.Photo KitAlysis Operating InstructionsFeatures:Designed and tested by synthetic chemists.Controller provides repeatable milliamp selection for photon intensity (sold seperately)527 nm wavelength0-30 mA variable LED outputNon-magnetic LED baseChemically resistant LED coverPTFE coated cablingPhoto Kitalysis Starter Kitrequired for operation (sold separately). Best when used withKitAlysis Benchtop Inertion Box(sold separately)... Read More |