| Description | NAD Kinase (NADK, EC 2.7.1.23) is widely present in animals, plants, microorganisms, and cultured cells. It is the only known enzyme that catalyzes the phosphorylation of NAD⁺ to NADP⁺ in vivo. It can utilize ATP or inorganic polyphosphate [poly(P)] as a phosphoryl donor to catalyze the NAD Kinase (NADK, EC 2.7.1.23) is widely present in animals, plants, microorganisms, and cultured cells. It is the only known enzyme that catalyzes the phosphorylation of NAD⁺ to NADP⁺ in vivo. It can utilize ATP or inorganic polyphosphate [poly(P)] as a phosphoryl donor to catalyze the phosphorylation of NAD(H), generating NADP(H). Therefore, NADK plays a crucial role in synthesizing NADP(H) and regulating the balance between NAD(H) and NADP(H).Assay PrincipleNADK catalyzes the phosphorylation of NAD⁺ to generate NADP⁺. The generated NADP⁺ is then reduced to NADPH by Glucose-6-Phosphate Dehydrogenase (G6PDH). The rate of increase in NADPH, measured by the rise in absorbance at 340 nm, reflects the activity of NADK.Component50TStorageExtraction Buffer50 mL2-8℃Reagent 125 mL2-8℃Reagent 250 mL2-8℃Reagent 31EA-20℃Reagent 41EA-20℃Required Materials and Equipment (Not Provided)UV spectrophotometer, benchtop centrifuge, adjustable pipettes, 1 ml quartz cuvette, mortar and pestle, ice, and distilled water.Sample Preparation1.Bacteria, Cells, or Tissues:Bacteria or Cultured Cells: Collect cells by centrifugation and discard the supernatant. Add Extraction Buffer at a ratio of 1 ml per 5-10 million cells (e.g., 1 ml for 5 million cells). Sonicate on ice (20% power or 200W, pulse 3s on/10s off, repeat 30 times). Centrifuge at 8000 g, 4°C for 10 min. Collect the supernatant and keep it on ice.Tissues: Homogenize tissue on ice in Extraction Buffer at a ratio of 1:5-10 (w/v) (e.g., 0.1 g tissue in 1 ml buffer). Centrifuge the homogenate at 8000 g, 4°C for 10 min. Collect the supernatant and keep it on ice.2.Serum (or Plasma) Samples: Assay directly.Assay Procedure:1.Preheat the spectrophotometer for at least 30 min. Set wavelength to 340 nm. Zero with distilled water.2.Pre-warm Reagent 1 and Reagent 2 at 37°C (for mammalian samples) or 25°C (for other species) for at least 15 min.3.Working Solution I Preparation: Add 12 mL of Reagent 1 to the contents of Reagent 3. Mix thoroughly. Aliquot and store unused portions at -20°C. Avoid repeated freeze-thaw cycles.Working Solution II Preparation: Add 45 mL of Reagent 2 to the contents of Reagent 4. Mix thoroughly. Aliquot and store unused portions at -20°C. Avoid repeated freeze-thaw cycles.4.Assay Setup:ReagentTest Tube (µL)Control Tube (µL)Sample100100Working Solution I400Reagent 1400Mix thoroughly. Incubate at 37°C (mammalian) or 25°C (other species) for 15 min.Immediately boil for 2 min (tighten caps to prevent evaporation).Cool on ice.Centrifuge at 10,000 g, 25°C for 10 min. Collect the supernatant.5.Detection:ReagentVolume (µL)Supernatant (from step 4)200Working Solution II800Add reagents to a new tube or cuvette. Mix thoroughly after addition.Let the reaction stand at room temperature for 15 min.Measure the absorbance at 340 nm.Calculate ΔA = ATest - AControl.NADK Activity Calculation:General Parameters:VTotal (Total reaction volume for detection step) = 5 × 10⁻⁴ L (0.5 mL = 500 µL)ε (NADPH molar extinction coefficient) = 6.22 × 10³ L/mol/cmd (Cuvette light path) = 1 cmVSample (Sample volume in initial reaction) = 0.1 mL (100 µL)VSample Total (Total extraction volume) = 1 mLT (Reaction time for NADK enzyme step) = 15 minCpr (Sample protein concentration, mg/mL)W (Sample mass, g)500 (Cell/Bacteria count in millions for example calculation: 5 million)1. For Serum (Plasma):Definition: One unit of activity is defined as the amount of enzyme that generates 1 nmol of NADP⁺ per minute per ml of serum.Calculation:NADK Activity (nmol/min/ml) = [ΔA × VTotal ÷ (ε × d) × 10⁹] ÷ VSample ÷ TSimplified Formula: NADK (nmol/min/ml) = 53.59 × ΔA2. For Tissues, Bacteria, or Cells:a. Based on Sample Protein Concentration:Definition: One unit of activity is defined as the amount of enzyme that generates 1 nmol of NADP⁺ per minute per mg of protein.Calculation:NADK Activity (nmol/min/mg prot) = [ΔA × VTotal ÷ (ε × d) × 10⁹] ÷ (VSample × Cpr) ÷ TSimplified Formula: NADK (nmol/min/mg prot) = 53.59 × ΔA ÷ Cprb. Based on Sample Fresh Weight:Definition: One unit of activity is defined as the amount of enzyme that generates 1 nmol of NADP⁺ per minute per gram of fresh tissue.Calculation:NADK Activity (nmol/min/g fresh weight) = [ΔA × VTotal ÷ (ε × d) × 10⁹] ÷ (W × VSample / VSample Total) ÷ TSimplified Formula: NADK (nmol/min/g fresh weight) = 53.59 × ΔA ÷ Wc. Based on Bacterial or Cell Density:Definition: One unit of activity is defined as the amount of enzyme that generates 1 nmol of NADP⁺ per minute per 10⁴ cells.Calculation (example for 5 million cells in 1 ml extract):NADK Activity (nmol/min/10⁴ cell) = [ΔA × VTotal ÷ (ε × d) × 10⁹] ÷ (500 × VSample / VSample Total) ÷ TSimplified Formula: NADK (nmol/min/10⁴ cell) = 0.107 × 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 introduction：Product introduction：Cell Cycle Assay Kit Plus ( Cell Cycle Assay Kit Plus ) has certain applicability for live cells and fixed cell cycle detection. For different types of cells, whether it is applicable or not needs to be determined after testing. Cell Cycle Product introduction：Product introduction：Cell Cycle Assay Kit Plus ( Cell Cycle Assay Kit Plus ) has certain applicability for live cells and fixed cell cycle detection. For different types of cells, whether it is applicable or not needs to be determined after testing. Cell Cycle Assay Kit Plus ( Cell Cycle Assay Kit Plus ) uses RedNucleus I staining to detect cell cycle. RedNucleus I is a far-infrared nucleic acid dye with cell membrane permeability, which can quickly enter living cells, specifically bind to DNA, and perform cell cycle detection on living cells without RNase digestion. Compared with the traditional PI staining method, the cells do not need to be broken or fixed, and the operation is simpler. RedNucleus I is a fluorescent dye of double-stranded DNA, and the fluorescence intensity after binding to double-stranded DNA is proportional to the content of double-stranded DNA. The intracellular DNA content can be measured by flow cytometry, and then the cell cycle analysis can be carried out according to the distribution of DNA content. After RedNucleus I staining, assuming that the fluorescence intensity of G0 / G1 phase cells is 1, the theoretical value of the fluorescence intensity of G2 / M phase cells containing two copies of genomic DNA is 2, and the fluorescence intensity of S phase cells undergoing DNA replication is between 1-2. In addition, RedNucleus I is compatible with dyes such as Horizon BV / BUV, FITC and R-PE, and can be periodically detected after sample staining.The kit is usually used to detect the cell cycle of cultured adherent or suspended cells. If it is used for cell cycle detection of tissues, the tissues must be digested into a single cell state.Matters needing attention：1. please centrifuge the product to the bottom of the tube immediately before use, and then conduct subsequent experiments. 2. this product is applicable to the detection of living cells and fixed cell cycle with certain limitations. Whether it is applicable to different types of cells needs to be determined after testing. If fixation is needed, it is recommended to use ice bath pre cooling 75-80% ethanol -20 ℃ to fix cells overnight. 3. fluorescent dyes have quenching problems. Please try to avoid light during storage and use to slow down fluorescence quenching. 4. for your safety and health, please wear experimental clothes and disposable gloves.Instruction： Experimental materials ( self-provided )：①cell lines or other cell samples ( self-prepared ) ；②This kit ; ③ trypsin ( self-prepared ) ;④ Cell culture medium containing FBS ( self-prepared ) ; Experimental procedure： 1.Preparation of cell samples : ( 1 ) ( This step is for adherent cells, if suspended cells, can be carried out directly step ( 2 ) ) Digest cells with trypsin, add cell culture medium, gently blow away cells, collected into the centrifuge tube. Note : The number of cells on the machine needs to reach 50,000 and above, so the initial number of cells collected needs to be sufficient. ( 2 ) Centrifuged about 1000 g for 3-5 min to precipitate cells. Carefully remove the supernatant, add about 1 mL of ice bath pre-cooled 1 × staining buffer ( 10 × staining buffer diluted with diH2O at 1 : 10 ), re-suspend the cells. Repeat once. ( 3 ) Centrifuged about 1000 g for 3-5 min to precipitate cells. After the supernatant was discarded, 1 mL of culture medium was added to re-suspend the cells ( for fixed cells, 1 × PBS can also be used to re-suspend ). Gently flick the bottom of the centrifuge tube to properly disperse the cells to avoid cell aggregation. 2.Staining : 4 µL of RedNucleus I staining solution was added to each tube of cell samples, slowly and fully mixed, and incubated at room temperature in dark for 20 min ( or incubated at 37 ° C in dark for 5-10 min ). The optimal incubation time of different cells is different, and the staining time can be adjusted and optimized according to the actual staining effect to obtain a more ideal staining effect. 3.Flow cytometry detection and analysis : Excited at 638 nm by flow cytometry, it is recommended to detect in RL3 or FL4 channels, or use RL1 and RL2 channels. Cell DNA content analysis and light scattering analysis were performed using appropriate analysis software.Scope of application：Cell cycle detection... Read More | Inquire | Product content: M665794Component125 TStorageM665794A2×miRNA qPCR Mixture (ROX)2×750 µL-20℃. Avoid freeze/thaw cycleM665794BReverse Primer, 10 µM60 µL-20℃. Avoid freeze/thaw cycleM665794CddH2O1.5 mL-20℃. Avoid freeze/thaw cycle Product Introduction:This kitProduct content: M665794Component125 TStorageM665794A2×miRNA qPCR Mixture (ROX)2×750 µL-20℃. Avoid freeze/thaw cycleM665794BReverse Primer, 10 µM60 µL-20℃. Avoid freeze/thaw cycleM665794CddH2O1.5 mL-20℃. Avoid freeze/thaw cycle Product Introduction:This kit uses the principle of SYBR Green I chimeric fluorescent dye method for miRNA fluorescence quantitative PCR detection. The kit includes 2 x miRNA qPCR Mixture and Reverse Primer required for detection. 2 x miRNA qPCR Mixture is a new generation pre mixed form of fluorescence quantitative PCR detection reagent specially developed for miRNA quantitative detection. The fluorescent dye SYBR Green I contained in it can bind to all double stranded DNA, making the product suitable for detecting different target sequences without the need to synthesize specific labeled probes. The GoldStar Taq DNA polymerase is a chemically modified and highly efficient thermal starter enzyme, coupled with a unique buffer system, which enhances reaction specificity, sensitivity, and enables accurate quantification of miRNA over a wider range. The 2x miRNA qPCR Mixture contains ROX dye and is suitable for fluorescence quantitative PCR instruments that require ROX as a calibration dye.Note: This kit must be used in conjunction with the miRNA cDNA first strand synthesis kit.Self prepared experimental materials: qPCR upstream primer.Forward Primer design principles:1. Follow the most common principles of primer design.2.Based on mature miRNA sequences, replacing U with T is the most basic and simplest design method.3.The Tm value of the downstream primer provided in the reagent kit is 63.6 ℃, and the Tm value of the upstream primer should be designed to be around 63.6 ℃ as much as possible.4. If the Tm value of the primer directly designed according to principle "2" is too low, several bases (preferably G or C bases) can be added to the 5 'end of the primer; One or several A bases can also be added at the 3 'end; Alternatively, both the 5 'and 3' ends can be modified simultaneously.5.If the Tm value of a primer designed directly according to principle "2" is too high, several bases can be removed from the 5 'or 3' end of the primer.Notes:1. Before using the reagent, please gently mix it upside down to avoid foaming, and use it after a brief centrifugation.2. The amount of miRNA first strand cDNA added should not exceed 10% of the volume of Real time PCR.3. For special detection systems, high content of cDNA templates can easily lead to non-specific amplification. Dilute cDNA appropriately (10 or 100 times dilution) based on the abundance of detected miRNAs.4. The 2x miRNA qPCR Mixture in this product contains SYBR Green I and ROX dyes. When storing this product or preparing PCR reaction solution, strong light exposure should be avoided.5. Avoid repeated freezing and thawing of this product. Repeated freezing and thawing may cause a decrease in product performance. This product can be stored at -20 ℃ for long-term storage. If frequent use is required in the short term, the 2xmiRNA qPCR Mixture can be stored at 2-8 ℃. However, the Reverse primer still needs to be stored at -20 ℃.Operation steps:1. Melt 2 x miRNA qPCR Mixture and Reverse Primer at room temperature (10 µ M). 2. When using, please gently mix the 2x miRNA qPCR Mixture upside down to avoid foaming, and use after brief centrifugation. If the reagent is not well mixed, its reaction performance will decrease.3. Place the reagent on ice and prepare the reaction system according to the following table: reagent volume final concentration 2×miRNA qPCR Mixture（ROX） 10 µl 1× Forward primer（10 µM） 0.4µl 0.2 µM Reverse primer（10 µM） 0.4µl 0.2 µM MiRNA first strand cDNA X µl — ddH2O up to 20 µl —4. The reaction program is set as follows:Attention!The pre denaturation reaction of this product must be completed at 95 ℃ for 10 minutes! Note: 1) The hot start enzyme used in this product must be activated under pre denaturation conditions of 95 ℃ and 10 minutes.2) The annealing temperature should be set at 60-64 ℃ as a reference range. When non-specific reactions occur, the annealing temperature can be increased... Read More | N665925 Component 1 mL 5 mL Storage N665925A 2×SYBR qPCR Master Mix 1 1 mL 5 mL -20℃. Avoid freeze/ Thaw cycle. N665925B qPCR Primer Mix 1 100 µL 500 µL -20℃. Avoid freeze/ Thaw cycle. N665925C DNA Standard I 100 µL 500 µL -20℃. Avoid freeze/ Thaw cycle. N665925 Component 1 mL 5 mL Storage N665925A 2×SYBR qPCR Master Mix 1 1 mL 5 mL -20℃. Avoid freeze/ Thaw cycle. N665925B qPCR Primer Mix 1 100 µL 500 µL -20℃. Avoid freeze/ Thaw cycle. N665925C DNA Standard I 100 µL 500 µL -20℃. Avoid freeze/ Thaw cycle. N665925D DNA Standard II 100 µL 500 µL -20℃. Avoid freeze/ Thaw cycle. N665925E DNA Standard III 100 µL 500 µL -20℃. Avoid freeze/ Thaw cycle. N665925F DNA Standard IV 100 µL 500 µL -20℃. Avoid freeze/ Thaw cycle. N665925G DNA Standard V 100 µL 500 µL -20℃. Avoid freeze/ Thaw cycle. N665925H 50×High ROX 40 µL 200 µL -20℃. Avoid freeze/ Thaw cycle.Product IntroductionThis product is a real-time fluorescence quantitative PCR of the products after NGS library construction using a dye method (SYBR Green I).(qPCR). The kit provides the reaction mixes, DNA primer mixtures, standards and sample dilutions required for the qPCR process, making the reagent system complete and easy to use. The fluorescent dye SYBR Green I contained in the reaction mixture can bind to all double-stranded DNA; the GoldStar Taq DNA Polymerase used is a chemically modified new high-efficiency hot-start polymerase, and the activation of the enzyme needs to be incubated at 95℃ for 10 minutes. the product has high specificity, high amplification efficiency, and is able to quickly and accurately quantify the concentration of the constructed libraries. The product is highly specific and efficient in amplification, and can quickly and accurately quantify the concentration of the constructed library.ROX dye is used to correct the fluorescence signal error generated between wells of a quantitative PCR instrument, and is generally used in Real Time PCR amplifiers from ABI, Stratagene, and other companies. The excitation optics vary from instrument to instrument, so the concentration of ROX dye must be matched to the corresponding fluorescence quantitative PCR instrument.Instruments that do not require ROX calibration: Roche LightCycler 480, Roche LightCyler 96, Bio-rad iCyler iQ, iQ5, CFX96, etc.Instruments requiring Low ROX calibration: ABI Prism7500/7500 Fast, QuantStudio®3 System, QuantStudio®5 System, QuantStudio®6 Flex System, QuantStudio®7 Flex System, ViiA 7 System, Stratagene Mx3000/Mx3005P, Corbett Rotor Gene 3000, and others.Instruments requiring High ROX calibration: ABI Prism7000/7300/7700/7900, Eppendorf, ABI Step One/Step One Plus, etc.Note: High Rox and Low Rox are formulated as described in Use 2.Scope of applicationThis product is designed for absolute quantification of the concentration of Ion torrent platform second generation sequencing libraries. The end of the library contains Ion torrent P5 and P7 microarray binding sequences, the length of which does not exceed 1kb, and the concentration is not less than 0.005pM can be used to perform quantitative experiments with this product. The qPCR Primer Mix provided in the kit contains the following two primer sequences:Primer 1:5'-CCA TCT CAT CCC TGC GTG TC - 3' Primer 2: 5'-CCT CTC TAT GGG CAG TCG GTG AT-3'The primer sequence can be used in advance to confirm whether the library can be amplified by that primer pair.Usage1. Amplification template preparationThe library samples to be detected were diluted with TE (10 mM Tris-Cl, pH 8.0, 1 mM EDTA), and the concentration after dilution was as close as possible to the range of 0.05-50 pM. 4°C on ice was set aside.2. qPCR reaction system preparationThe desired cryopreservation reagent is pre-melted completely and mixed by inverting several times before preparation, then centrifuged briefly and set aside.The base reaction system for 20 µl was as follows:Reagent20 µl Reaction system2×SYBR qPCR MasterMix10 µlqPCR Primer Mix 10.8 µlTemplate4 µlddH₂O5.2 µlDescription: High Rox model: add 1 µl High Rox per 50 µl of reaction system;Low Rox model: 1 µl High Rox per 500 µl of reaction system.Prepare a sufficient amount of reaction system mixture according to the need, mix well and add to the reaction wells in a volume of 16 µl per well, add the same volume of TE to the blank control, and then add the prepared standards and diluted samples to the corresponding reaction wells in a volume of 4 µl/well. It is recommended to use 20 µl reaction system, if you need to carry out a smaller system reaction, the system components can be reduced in equal proportion.3. qPCR reaction program1) Please use 60-64℃ as a reference for setting range of annealing temperature, and increase the annealing temperature when non-specific reaction occurs.2) If the average length of the library is greater than 700bp, the annealing/extension time should be increased appropriately.data analysis1. Standard curve productionThe standard curve was plotted using Ct values in the valid range. The standard curve correlation coefficient R2 should not be less than 0.99 and the slope should lie between -3.1 and -3.6. If the standard curve parameters are not reasonable, it is recommended to repeat the experiment.DNA Standard NameDNA Standard ConcentrationDNA Standard I50 pMDNA Standard II5 pMDNA Standard III0.5 pMDNA Standard IV0.05 pMDNA Standard V0.005 pM2. Library concentration calculationThe difference in Ct between the three replicate wells of the experiment should be no more than 0.2, otherwise the invalid data should be deleted or the experiment should be repeated. Do not use the Ct outside the valid Ct range of the standard curve to calculate the concentration of the diluted libraries. Please refer to the data processing Excel of this product for the specific library concentration calculation method.matters needing attention1. Before testing, these instructions should be read in detail. It should be operated by personnel with professional experience or qualified by training.2. For use, please mix gently by turning up and down, avoid foaming as much as possible, and use it after centrifugation for a short period of time.3. Avoid repeated freezing and thawing of the product, repeated freezing and thawing may degrade the performance of the product.4. When preparing the reaction solution, please use new or non-contaminated tips and centrifuge tubes to prevent contamination as much as possible... Read More |