| 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 | The bacterial viability / toxicity detection kit contains two fluorescent dyes. Nucgreen is a green nucleic acid dye that can stain live and dead bacteria; Ethd III is a red nucleic acid dye that only stains dead bacteria with damaged cell membranes. When nucgreen and ethd III are properly mixed, The bacterial viability / toxicity detection kit contains two fluorescent dyes. Nucgreen is a green nucleic acid dye that can stain live and dead bacteria; Ethd III is a red nucleic acid dye that only stains dead bacteria with damaged cell membranes. When nucgreen and ethd III are properly mixed, the bacteria with intact cell membrane appear green, while the bacteria with damaged cell membrane can appear green and red under different channels, respectively. A common criterion for bacterial viability is the ability to propagate in a suitable nutrient medium, known as a growth assay. This kit is generally in good agreement with the growth assay results in liquid or solid medium. However, under certain conditions, membrane damaged bacteria may recover and propagate in nutrient medium, and such bacteria will be identified as dead bacteria in this assay. On the contrary, some bacteria with intact membranes may not be able to propagate in nutrient medium, but will be recognized as viable bacteria in this assay. Therefore, if there is a large difference between the test results of this kit and the bacterial growth assay, the above possibilities should be considered. Component： Product parameters： NucGreen: Ex/Em = 503/530 nm (结合 DNA)；EthD-III: Ex/Em = 530/620 nm (结合 DNA)。Usage:1 Preparation of control samples for live and dead bacteria (optional)1. Cultivate 4 mL of bacteria in liquid medium until late logarithmic phase.2. Prepare two 1 mL bacterial solutions in an EP tube and centrifuge for 10-15 minutes under 5000-10000 g conditions.3. Remove the supernatant and add 0.3 mL of 0.85% NaCl resuspended bacteria to one of the EP tubes, and 1 mL of 0.85% NaCl resuspended bacteria to the other tube.4. Add 0.7 mL of isopropanol to a tube containing 0.3 mL of 0.85% NaCl, and mix thoroughly (with a final concentration of 70% isopropanol) to prepare a dead bacterial sample.5. Incubate the two samples at room temperature for 1 hour and mix every 15 minutes.6. Centrifuge the two samples at 5000-10000 g for 10-15 minutes.7. Remove the supernatant, add 1 mL of 0.85% NaCl to resuspend the bacteria in both samples, and centrifuge again as in step 6.8. Use a spectrophotometer to measure the absorbance values (OD670) of two bacterial suspensions at 670 nm.9. Adjust the density of the two bacterial suspensions (live and dead) to 108 bacteria/mL (OD670 ≈ 0.3), and then dilute with 0.85% NaCl at 1:100 to achieve a final density of 106 bacteria/mL.10. Mix two bacterial suspensions as shown in the table below to obtain the required live cell ratio: dead cell ratio.Table 1 Mix live and dead bacterial suspensions by a certain volume to achieve the required ratio of live and dead cellsLive cells: Dead cellsVolume of viable bacterial suspension（mL）Volume of dead bacterial suspension（mL）0:10001.010:900.10.920:800.20.830:700.30.750:500.50.5100:01.00II Staining methods for fluorescence microscopy observation1. Mix 1 volume of component A, NucGreen, and 2 volumes of component B, EthD-III, in a microcentrifuge tube. After thorough mixing, add 8 volumes of 0.85% NaCl solution to obtain a 100 x dye solution.2. Every 100 µ L bacterial suspension, add 1 µ 100 x dye solution of L.3. Mix thoroughly and incubate at room temperature in the dark for 15 minutes.4. Take 5 µ The bacterial suspension after L staining was dropped onto a glass slide with an 18 mm square cover glass.5. Observe under a fluorescence microscope. The fluorescence of live and dead bacteria can be observed simultaneously under any standard FITC long-acting filter. Alternatively, live (green fluorescent) and dead (red fluorescent) bacteria can be observed using FITC and Cy3 (or Texas Red) channels, respectively.Attention: (1) Before staining bacteria, attention must be paid to removing residues of growth media. Nucleic acid and other media components can bind to NucGreen and EthD-III dyes in some way, resulting in unacceptable staining changes. A simple washing step is usually sufficient to remove interfering media components from bacterial suspension. It is not recommended to use phosphate buffer solutions as they can reduce staining efficiency. (2) Before starting the formal experiment, the dye concentration should be adjusted to distinguish between NucGreen labeling live bacteria and EthD-III labeling dead bacteria. The optimal concentration may vary depending on the bacterial strain. It is generally best to use the lowest dye concentration that can provide sufficient signal. The above conditions have been optimized for staining live/dead cells of Escherichia coli.III Before starting the staining method experiment of flow cytometry, please read the precautions under the fluorescence microscope staining steps.According to Table 1, add 11 different proportions of live and dead bacteria to the EP tube. Each of the 11 samples has a volume of 1 mL.2. Add 12 µ The A component of L, NucGreen, and 24 µ The B component EthD-III of L was mixed in a microcentrifuge tube. Add 3 to each of the 11 samples µ Mix the mixed dyes of L thoroughly by blowing them up and down several times. (Note: Additional control bacterial samples need to be prepared for separate NucGreen and EthD-III staining)3. Incubate at room temperature in the dark for 15 minutes.4. Analyze each sample using a flow cytometer, detect NucGreen positive cells using FITC channels, and detect EthD-III positive cells using PI or PE channels.Matters needing attention：1. please centrifuge the product to the bottom of the tube immediately before use, and then conduct subsequent experiments. 2. if the orifice plate is used for detection, a small amount of bacterial liquid can be left for imaging after standing for 10 min, which can effectively reduce the background. 3. in order to be closer to the real results, it is recommended to keep the brightness of red fluorescence consistent with that of green fluorescence in merge pictures. 4. fluorescent dyes have quenching problems. Please try to avoid light during experimental operation to slow down fluorescence quenching. 5. for your safety and health, please wear experimental clothes and disposable gloves.Scope of application：Staining of dead and live bacteria... Read More | Calcium, the most abundant mineral in the human body, is a crucial intracellular element that is responsible for regulating many physiological and pathological processes. Calcium is found in either the free ion form or in bound complexes, for example the calcium phosphate and calcium carbonate Calcium, the most abundant mineral in the human body, is a crucial intracellular element that is responsible for regulating many physiological and pathological processes. Calcium is found in either the free ion form or in bound complexes, for example the calcium phosphate and calcium carbonate complexes that make up bone tissue. Numerous physiological processes, including muscle contraction, cell adhesion, hormones/ neurotransmitters release, glycogen metabolism, cell proliferation/differentiation, blood clotting, nerve or synapthetic impulse transmission, and structural support of the skeleton are regulated by calcium signaling. Defects in the integrity of cell-specific calcium signaling systems may be associated with certain human diseases.Calcium Colorimetric Assay kit has been used to measure calcium concentration in hippocampal samples and MC3T3-E1 mouse osteoblast cell line, which were cultured in osteogenic induction medium... Read More | DescriptionThe 200 nm Coupling Kit makes conducting lateral flow tests and biomolecule separation (including cell separation) easier and more flexible. The Kit contains AnteoBind™activated 200 nm magnetic particles that give you increased antibody binding capacity and functionality, while the DescriptionThe 200 nm Coupling Kit makes conducting lateral flow tests and biomolecule separation (including cell separation) easier and more flexible. The Kit contains AnteoBind™activated 200 nm magnetic particles that give you increased antibody binding capacity and functionality, while the included blocking buffer decreases background noise.Reduce reagent preparation time; remove traditional surface preparation steps such as EDC and replace these steps with the 200 nm pre-activated magnetic particles provided. This Kit reduces aggregation and gives you the freedom and ability to produce multifunctional particles for diverse applications, including dual labelling.For lateral flow tests, magnetic particles are easier to handle than gold. Magnetic separation removes the need to perform centrifugation and filtration concentration. Magnetic particles can provide greater sensitivity than gold during lateral flow tests.Binding Capacity and Polydisperity IndexBinding Capacity: > 50 µg IgG/mgPolydispersity Index (PdI)*: < 0.3* The Polydispersity Index (PdI) is dimensionless and determined using Dynamic Light Scattering (DLS). The PdI is scaled such that values smaller than 0.05 are rarely seen and values greater than 0.7 indicate that the sample has a very broad size distribution and poor monodispersity.Particle based Immunoassays, Lateral Flow, Bioseparations and Immunoprecipitation... Read More | Product content： Component S665549 50 preps Buffer SW 60 ml Buffer SL 60 ml Buffer GL 50 ml Buffer GW1（concentrate） 2X13 ml Buffer GW2（concentrate） 15 ml Buffer GE 15 ml Spin Columns DM 50 with Collection Tubes 50Product IntroductionThis kit is suitable for Product content： Component S665549 50 preps Buffer SW 60 ml Buffer SL 60 ml Buffer GL 50 ml Buffer GW1（concentrate） 2X13 ml Buffer GW2（concentrate） 15 ml Buffer GE 15 ml Spin Columns DM 50 with Collection Tubes 50Product IntroductionThis kit is suitable for extracting total DNA from fecal samples, including the total DNA of cells, bacteria, parasites, and viruses in the samples, as well as samples containing high concentrations of PCR reaction inhibitors. This product can process up to 300 mg of fecal samples and purify to obtain mainly 20-30 kb DNA fragments. The purification process does not require toxic solvents such as phenol or chloroform, and does not require ethanol precipitation. High purity DNA can be obtained within one hour. This reagent kit adopts a unique buffering system to efficiently bind DNA from the lysis solution to the adsorption column. At the same time, protein impurities and other organic compounds that inhibit downstream reactions in feces can flow through the membrane. Inhibitors of PCR and enzyme reactions, as well as residual impurities, can be effectively removed through two washing steps. Finally, high-purity DNA can be obtained by washing with low salt buffer or water. The purified DNA can be directly used for downstream experiments such as enzyme digestion, PCR, Real Time PCR, library construction, Southern Blot, and molecular labeling.Preparation and important precautions before the experiment1. Samples should avoid repeated freeze-thaw cycles, otherwise it may result in smaller extracted DNA fragments and a decrease in extraction volume.2.Before the first use, anhydrous ethanol should be added to Buffer GW1 and GW2 according to the instructions on the reagent bottle label.3. Before use, please check whether there is crystallization or precipitation in Buffer SL and Buffer GL. If there is crystallization or precipitation, please dissolve Buffer SL and Buffer GL again in a 56 ℃ water bath.4. If downstream experiments are sensitive to RNA contamination, 4 can be added after adding Buffer SL µ RNase A of DNase Free (100 mg/ml) is not provided in this kit. If needed, it can be ordered separately from our company, item number: S665549Operation steps1. Take a fecal sample of 100-300 mg and place it in a centrifuge tube (provided by oneself).2. Add 1 ml of Buffer SW and vortex for 3-5 minutes to evenly disperse the sample in the solution. Centrifuge at 12000 rpm (~13400 × g) for 1 minute and discard the supernatant.3. Add 1 ml of Buffer SL and vortex for 3-5 minutes to evenly disperse the sample in the solution. Take a water bath at 65 ℃ for 20 minutes and vortex for 15 seconds every 5 minutes. Note: To remove RNA, add 4 after completing the above steps µ RNase A solution (product number: CW0601S) with a concentration of 100 mg/ml, shake well and let stand at room temperature for 5-10 minutes.4.Centrifuge at 2000 rpm for 3 minutes and transfer the supernatant to a new centrifuge tube (provided by oneself).5. Add an equal volume of Buffer GL to the supernatant, invert and mix 15-25 times, and leave on ice for 5 minutes. Centrifuge at 12000 rpm for 5 minutes. Attention: At this time, the liquid may be in a transparent or turbid state, which does not affect the experiment. 6. Add the supernatant obtained in step 5 to the spin columns DM that have been loaded into the collection tube. If the solution cannot be added at once, it can be transferred multiple times. Centrifuge at 12000 rpm (~13400 × g) for 1 minute, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube.7. Add 500 to the adsorption column µ Buffer GW1 (check if anhydrous ethanol has been added before use), centrifuge at 12000 rpm for 1 minute, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube. 8. Repeat step 7.9. Add 500 to the adsorption column µ Buffer GW2 (check if anhydrous ethanol has been added before use), centrifuge at 12000 rpm for 1 minute, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube.10.12000 rpm for 2 minutes and discard the waste liquid from the collection tube. Place the adsorption column at room temperature for a few minutes to thoroughly air dry. Note: The purpose of this step is to remove residual ethanol from the adsorption column, which can affect subsequent enzymatic reactions (such as enzyme digestion, PCR, etc.).11. Place the adsorption column in a new centrifuge tube (self provided) and add 50-100 drops of suspended droplets to the middle of the adsorption column µ L Buffer GE or sterilized water, leave at room temperature for 2-5 minutes, centrifuge at 12000 rpm for 1 minute, collect DNA solution, and store DNA at -20 ℃.Note: 1) If downstream experiments are sensitive to pH or EDTA, they can be washed off with sterilized water. The pH value of the eluent has a significant impact on the elution efficiency. If water is used as the eluent, its pH value should be ensured to be between 7.0-8.5 (NaOH can be used to adjust the pH value of the water to this range). When the pH value is below 7.0, the elution efficiency will be reduced2) Incubating at room temperature for 5 minutes before centrifugation can increase yield.3) Use an additional 50-100 µ Further washing with buffer GE or sterilized water can increase yield.4) If you want to increase the final concentration of DNA, you can add the DNA eluent obtained in step 11 back onto the adsorption membrane and repeat step 11; It is possible to increase the final concentration of DNA, but it may reduce the total yield. If the amount of DNA is less than 1 µ g. Recommended 50 µ Wash with buffer GE or sterilized water.5) DNA stored in water can be affected by acidic hydrolysis. If long-term storage is required, it is recommended to elute with Buffer GE and store at -20 ℃.6) The residual trace PCR inhibitors in the genomic DNA template may have adverse effects on the PCR reaction, which can be solved by diluting DNA by 2-10 times... Read More |