| 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 | Product content C665709Component50 TStorageC665709ABuffer CL45 mLRTC665709BBuffer CB (concentrate)60 mLRTC665709CBuffer GW1 (concentrate)13 mLRTC665709DBuffer GW2 (concentrate)15 mLRTC665709EBuffer EBL10 mLRTC665709FProteinase K100 mgRTC665709GProteinase K Storage Buffer5 mLRTC665709HSpin Columns DFProduct content C665709Component50 TStorageC665709ABuffer CL45 mLRTC665709BBuffer CB (concentrate)60 mLRTC665709CBuffer GW1 (concentrate)13 mLRTC665709DBuffer GW2 (concentrate)15 mLRTC665709EBuffer EBL10 mLRTC665709FProteinase K100 mgRTC665709GProteinase K Storage Buffer5 mLRTC665709HSpin Columns DF with Collection Tubes50 EA2-8℃C665709ICentrifuge Tubes (L-1.5 mL)50 EART Product IntroductionThis kit is suitable for the extraction of free DNA from fresh or frozen serum, plasma, lymph fluid and other cell-free body fluids.This kit adopts centrifugal adsorption columns that can specifically bind nucleic acids and a unique buffer system.After the sample is lysed, the free DNA binds to the silica gel membrane under high salt conditions, and the free DNA elutes from the silica gel membrane at low salt and high pH. The product can handle liquid samples of 0.1-1 ml, and the elution volume of the configured high-efficiency micro adsorption column can be as low as 20 µl. The purified DNA is of high yield and quality, with maximum removal of proteins, pigments, lipids, and other inhibitors, and the rate of free DNA yield is highly dependent on the type of samples, storage conditions, time, and inter-individual variations. The quality of free DNA obtained from purification is stable and reliable, and can be directly used in molecular biology experiments such as PCR, fluorescence quantitative PCR and second generation sequencing.Self-contained reagents: anhydrous ethanol, isopropanol.Pre-experiment Preparation and Important NotesAdd 5 ml of Proteinase K Storage Buffer to Proteinase K to dissolve it and store it at -20℃. Do not leave the prepared Proteinase K at room temperature for a long time.Repeated freezing and thawing of the sample should be avoided, as this can lead to a decrease in extraction.This kit can extract 0.1-1 ml of liquid samples.Before use, please check Buffer CL, Buffer CB for crystallization or precipitation, if there is any crystallization or precipitation, please re-dissolve Buffer CL, Buffer CB by incubation at 56℃ in a water bath.Before first use isopropyl alcohol should be added to Buffer CB according to the instructions on the reagent bottle label, mixed well, and labeled on the reagent bottle label.Before the first use, anhydrous ethanol should be added to Buffer GW1 and Buffer GW2 according to the instructions on the label of the reagent bottle, mixed well, and labeled on the label of the reagent bottle.Preheat the water bath to 60°C before starting the experiment.The elution buffer Buffer EBL can be preheated to 60°C and used.Operation stepsAdd 20 µl of Proteinase K to the centrifuge tube (supplied).Add 200 µl of serum/plasma sample.Note: When the sample volume exceeds 200 µl, please increase the amount of Proteinase K, Buffer CL and Buffer CB reagents in equal proportions, and the specific amount of reagents added can be referred to the attached table.3. Add 160 µl Buffer CL, mix upside down and shake vigorously for at least 30 seconds.4. Incubate at 60°C for 30 minutes, during which time mixing was inverted several times.Note: Incubation of 200µl serum/plasma samples at 60°C for 10-15 minutes is sufficient.Add 360 µl of Buffer CB (check for addition of isopropanol before use) and shake until thoroughly mixed.Ice bath for 5 minutes and centrifuge briefly to concentrate the liquid on the walls and wall caps to the bottom of the tube.Add all of the solution obtained in step 6 to the adsorption columns (Spin Columns DF) that have been loaded into the collection tubes, and if the solution cannot be added all at once, it can be transferred in several times. centrifuge the columns at 12,000 rpm for 1 minute, pour off the waste solution from the collection tubes, and put the columns back into the collection tubes.Add 500µl of Buffer GW1 to the adsorbent column (check that anhydrous ethanol is added before use),centrifuge the column at 12,000rpm for 30 seconds, pour off the waste liquid in the collection tube, and put the adsorbent column back into the collection tube.Add 750 µl Buffer GW2 to the adsorbent column (check that anhydrous ethanol is added before use), centrifuge at 12,000 rpm for 30 seconds, pour off the waste liquid in the collection tube, and put the adsorbent column back into the collection tube.10. Add 750 µl of anhydrous ethanol to the adsorbent column and centrifuge at 12,000 rpm for 30 s. Pour off the waste liquid in the collection tube and put the adsorbent column back into the collection tube.11. Centrifuge at 12,000 rpm for 2 minutes and pour off the waste liquid in the collection tube. Leave the adsorption column at room temperature for several minutes to dry thoroughly.Note: The purpose of this step is to remove residual ethanol from the adsorption column, which can interfere with the subsequent enzymatic reaction.12. Place the adsorption column in a new centrifuge tube, add 20-100 µl Buffer EBL or sterilized water to the middle part of the adsorption column overhanging the column, leave it at room temperature for 2-5 minutes, centrifuge at 12,000 rpm for 1 minute, collect the DNA solution, and store the DNA at -20℃.Note: 1) If the downstream experiment is sensitive to pH, you can use sterilized water for elution. The pH value of the eluent has a great influence on the elution efficiency, if water is used as the eluent should ensure that its pH value is 7.0-8.5 (you can use NaOH to adjust the pH value of water to this range), and the elution efficiency is not high when the pH value is lower than 7.0.2) Preheat the elution buffer BufferEBL to 60℃ and use it, and incubate it at room temperature for 5 minutes before centrifugation to increase the yield.3) If the final concentration of DNA is to be increased, the resulting solution can be reintroduced into the adsorption column and left at room temperature for 2-5 minutes and centrifuged at 12,000 rpm for 1 minute.4) Because DNA preserved in water will be affected by acidic hydrolysis, for long-term storage, it is recommended to elute it with Buffer EBL and store it at -20℃.Table: Recommended reagent additions for different sample sizes... Read More | Inquire | Hydrogen peroxide, a reactive oxygen species produced through the metabolism of molecular oxygen, serves as both an intracellular signaling messenger and a source of oxidative stress. Hydrogen peroxide is generated in cells via multiple mechanisms such as the NOX-mediated ROS production by Hydrogen peroxide, a reactive oxygen species produced through the metabolism of molecular oxygen, serves as both an intracellular signaling messenger and a source of oxidative stress. Hydrogen peroxide is generated in cells via multiple mechanisms such as the NOX-mediated ROS production by neutrophils and macrophages (respiratory burst) or by the dismutase of superoxide anions produced as a result of electron leak during mitochondrial respiration. Abnormal hydrogen peroxide production contributes to oxidative cell damage and the progression of diseases such as asthma, atherosclerosis, osteoporosis, and neurodegeneration.Intracellular hydrogen peroxide assay kit has been used to measure intracellular hydrogen peroxide levels... Read More | Product content:M665754Component25 TStorageM665754ATris-HCl, 1 mM, PH 8.01 mL-20℃. Avoid freeze/thaw cycleM665754BE. coli Poly(A) Polymerase, 5 U/µL15 µL-20℃. Avoid freeze/thaw cycleM665754C10×Poly(A) Polymerase Buffer80 µL-20℃. Avoid freeze/thaw Product content:M665754Component25 TStorageM665754ATris-HCl, 1 mM, PH 8.01 mL-20℃. Avoid freeze/thaw cycleM665754BE. coli Poly(A) Polymerase, 5 U/µL15 µL-20℃. Avoid freeze/thaw cycleM665754C10×Poly(A) Polymerase Buffer80 µL-20℃. Avoid freeze/thaw cycleM665754DATP, 10 mM15 µL-20℃. Avoid freeze/thaw cycleM665754ERT Primer, 25 µM90 µL-20℃. Avoid freeze/thaw cycleM665754F5×SuperRT Buffer120 µL-20℃. Avoid freeze/thaw cycleM665754GUltraPure dNTP Mix, 10 mM each30 µL-20℃. Avoid freeze/thaw cycleM665754HSuperRT, 200 U/µL15 µL-20℃. Avoid freeze/thaw cycleM665754IRNase-Free Water1 mL-20℃. Avoid freeze/thaw cycle Product Introduction:This kit uses the method of adding a poly (A) tail at the 3 'end of miRNA to give miRNA a Poly (A) tail, followed by reverse transcription using Oligo (dT) - Universal tag universal reverse transcription primers to synthesize the first stranded cDNA corresponding to miRNA. The miRNA cDNA first strand synthesis kit contains all the reagents required for the miRNA 3 'end Poly (A) tail modification process and the reverse transcription process after modification. This kit has a very high Poly (A) modification and reverse transcription efficiency, which can range from 1 ng-2 µ The first strand of cDNA corresponding to miRNA was effectively obtained from the total RNA of g. And the operation is simple and fast, which can be used to simultaneously detect multiple miRNAs from a synthesized cDNA reaction. This not only reduces errors and saves samples, but also achieves high-throughput detection.Note: This kit must be used in conjunction with the miRNA fluorescence quantitative detection kit.Self prepared experimental materials: 1 ng-2 µ Total RNA of g, or 0.1 ng-1 µ Small molecule RNA of g.Notes:To prevent RNase pollution, attention should be paid to the following aspects:1. Use plastic products and gun heads without RNase to avoid cross contamination.2. Glassware should be dry baked at a high temperature of 180 ℃ for 4 hours before use. Plastic containers can be soaked in 0.5 M NaOH for 10 minutes, thoroughly rinsed with water, and then sterilized under high pressure.3. The solution should be prepared using water without RNase.4. Operators should wear disposable masks and gloves, and change gloves frequently during the experiment.Usage:A. The process of miRNA adding Poly (A) tail:1.based on the amount of RNA used, dilute the total RNA of 10 mM ATP with 1 mM Tris (pH 8.0) according to the following formula: ATP dilution coefficient=5000/__ ngExample: If the initial amount of total RNA is 100 ng, then the ATP dilution coefficient is 5000/100=50. About to dilute ATP 50 times (1 µ 10 mM ATP plus 49 for l µ 1 mM Tris at pH 8.0.2. Add the following reagents to the pre cooled RNase free reaction tube in the ice bath to a total volume of 25 µ L. reagent 25 µlReaction system final concentration total RNA* X µl Up to 2 µg 10×Poly(A) Polymerase Buffer 2.5 µl 1× Diluted ATP in step "1" 1 µl / E. coli Poly(A) Polymerase, 5U/µl 0.5 µl 2.5 U RNase-Free Water up to 25 µl /*The total RNA used in the reaction must contain small molecule RNA.This process can also directly use small molecule RNA (recommended dosage of 2-5) µ L. Please determine the amount added based on the abundance of the target miRNA.3. Gently mix the above reaction solution and briefly centrifuge to collect the liquid at the bottom of the tube. Incubate at 37 ℃ for 15 minutes. After this process is completed, immediately proceed with the synthesis of the first strand cDNA or temporarily store it at -20 ℃. If long-term storage is required, it is recommended to store at -80 ℃.B. The process of synthesizing the first strand of modified miRNA cDNA:1. Add the reagents in the table below to the pre cooled RNase free reaction tube in the ice bath until the final volume reaches 20µl: reagent 20 µlReaction system The above Poly (A) reaction solution 4 µl UltraPure dNTP Mix ，10 mM each 1 µl RT Primer ，25 µM 3 µl 5×SuperRT Buffer 4 µl SuperRT ，200 U/µl 0.5 µl RNase-Free Water 7.5 µl2. Gently mix the above reaction solution and briefly centrifuge to collect the liquid at the bottom of the tube. Incubate at 42 ℃ for 50 minutes.3.85 ℃ for 5 minutes and terminate the reaction. The synthesized cDNA reaction solution can be directly used for fluorescence quantitative detection experiments or stored at -20 ℃ for future use... Read More |