| Description | Lactic acid is an important intermediate product in the metabolic processes of organisms, closely related to carbohydrate metabolism, lipid metabolism, protein metabolism, and intracellular energy metabolism. Lactic acid concentration is a key indicator for evaluating glycogen metabolism and aerobicLactic acid is an important intermediate product in the metabolic processes of organisms, closely related to carbohydrate metabolism, lipid metabolism, protein metabolism, and intracellular energy metabolism. Lactic acid concentration is a key indicator for evaluating glycogen metabolism and aerobic metabolism. Abnormally high concentrations of lactic acid are associated with pathological conditions such as cancer, diabetes, and lactic acidosis.The detection principle of this kit is as follows: Lactate dehydrogenase catalyzes the conversion of L-lactate to pyruvate, simultaneously reducing NAD+ to NADH and H+. Further, through the hydrogen transfer action of 1-mPMS, WST-8 reacts to form a yellow, soluble formazan. The absorbance at 450 nm is measured to calculate the L-lactate content in the sample.Detection Range: 0.03-2 mMSensitivity: 0.03 mMApplicable Samples: Animal and plant tissues, cells, bacteria, serum (plasma), or other liquids.L1501211Component48T96TStorageL1501211ALactate Assay Buffer70 mL70 mL×22-8℃L1501211BLactate Dehydrogenase0.7 mL1.4 mL-20℃L1501211CLactate Dehydrogenase Cofactor0.5 mL1 mL-20℃L1501211DWST-8350 µL700 µL-20℃. Store in the dark.L1501211EEnhancer70 µL140 µL-20℃. Store in the dark.L1501211FL(+)-Lactate Standard (100 mM)50 µL100 µL-20℃Please check the quantity of each component before the experiment.An additional 10% of each component is provided beyond the specified volume for standard curve preparation or preliminary experiments.User-Provided Instruments and ReagentsTypeNameNotesInstrumentMicroplate ReaderCapable of measuring absorbance at 450 nm.Consumables96-well MicroplateStandard transparent plate.ReagentsPBS (pH 7.4)For washing samples.OthersHomogenizer (for tissue samples), incubator, ice machine, low-temperature centrifuge, adjustable pipettes and tipsUsing a multichannel pipette for large-scale detection can improve efficiency.Experimental Procedure1. Reagent PreparationReagent NameReagent PreparationPrecautionsLactate Assay BufferReady-to-use; equilibrate to room temperature before use.4℃保存 Store at 4°C.Lactate DehydrogenaseReady-to-use;Keep on ice during the experiment; store aliquots at -20°C.Lactate Dehydrogenase CofactorReady-to-use;Keep on ice during the experiment; store aliquots at -20°C.WST-8Ready-to-use;Keep on ice during the experiment; store aliquots at -20°C.EnhancerReady-to-use;Keep on ice protected from light during the experiment; store aliquots at -20°C protected from light.L(+)-Lactate Standard (100 mM)Equilibrate to room temperature before use.100 mM, store aliquots at -20°C.2. Standard PreparationAdd 20 µL of the 100 mM standard to 980 µL of Lactate Assay Buffer to prepare a 2 mM standard stock solution. Aliquot and store at -20°C for up to 6 months. Dilute the 2 mM standard stock solution sequentially with Lactate Assay Buffer to prepare standard working solutions with final concentrations of 1 mM, 0.5 mM, 0.25 mM, 0.125 mM, 0.0625 mM, and 0.03125 mM. Use Lactate Assay Buffer as the blank.Standard Working SolutionStandard (µL)Lactate Assay Buffer (µL)Concentration (mM)1200 µL of 2 mM022200 µL of 2 mM20013200 µL of 1 mM2000.54200 µL of 0.5 mM2000.255200 µL of 0.25 mM2000.1256200 µL of 0.125 mM2000.06257200 µL of 0.0625 mM2000.03125Blank020003. Sample PreparationNote: Fresh samples are recommended. If not used immediately, samples can be stored at -80°C for up to 1 month. NADH or NADPH present in cell or tissue extracts can create background for lactate assay. To remove NADH or NADPH background, an equal amount of sample can be assayed without lactate dehydrogenase, and the background reading should be subtracted from the lactate reading. Endogenous lactate dehydrogenase (LDH) can degrade lactate. Samples containing LDH (e.g., cell culture medium, cell or tissue lysates) should be processed using a 10 kDa MW cutoff ultrafiltration tube (centrifuge at 12,000 g, 4°C for 10 min; follow the filter instructions) to remove all proteins. Use the filtrate for detection, then store at -80°C.3.1 Animal/Plant Tissues: Weigh approximately 0.1 g of tissue sample, add 1 mL of Lactate Assay Buffer, and homogenize on ice. Centrifuge at 12,000 g, 4°C for 5 min. Transfer the supernatant to a new tube and keep on ice for detection.3.2 Cells or Bacteria: Collect 5×10^6 cells. Wash the cells or bacteria with pre-cooled PBS. Centrifuge at 800 g for 2 min, discard the supernatant. Add 1 mL of Lactate Assay Buffer, and disrupt using an ultrasonic homogenizer on ice for 5 min (power 20% or 200 W, ultrasonic 3 s, interval 7 s, repeat 30 times). Centrifuge at 12,000 g, 4°C for 5 min. Collect the supernatant and keep on ice for detection.3.3 Plasma and Serum (Other Biological Fluids): Detect directly.4. Experimental Steps4.1 Microplate Reader Preparation: Preheat for at least 30 minutes, set wavelength to 450 nm.4.2 Working Reagent Preparation: 50 µL of Working Reagent is required per well. To avoid loss, prepare for 55 µL per single well system: Pipette 31 µL Lactate Assay Buffer, 8 µL Lactate Dehydrogenase Cofactor, 5 µL WST-8, 1 µL Enhancer, and 10 µL Lactate Dehydrogenase. Mix well. The Working Reagent must be prepared freshly and used immediately.4.3 Assay System Setup: Set up the detection system in the microplate according to the table below. The standard curve generally needs to be performed only once.ReagentStandard Well (µL)Test Well (µL)Sample050Standard Working Solution500Working Reagent50504.4 Absorbance Measurement: Mix well and incubate at 37°C protected from light for 30 min. Read the absorbance at 450 nm, recorded as Ablank, Astandard, and Atest. 5. Result CalculationThe following provides both the derived formula and the simplified calculation formula, which are completely equivalent.5.1 Data ProcessingCalculate ΔAstandard= Astandard- Ablank, ΔAtest = Atest - Ablank.5.2 Standard Curve PlottingPlot the standard curve with standard concentration as the y-axis and ΔAstandard as the x-axis. Substitute ΔAstandard into the equation to obtain the y value (mM).5.3 Sample L-Lactate Content Calculation① Calculated based on sample weight:L-Lactate (µmol/g) = y × Vsample ÷ (W × Vsample ÷ Vtotal) × n = y ÷ W × n② Calculated based on cell or bacterial count:L-Lactate (µmol/10⁴ cells) = y × Vsample ÷ (500 × Vsample ÷ Vtotal) × n = y ÷ 500 × n③ Calculated based on liquid volume:L-Lactate (mM) = y × Vsample ÷ Vsample × n = y × n④ Calculated based on protein concentration:L-Lactate (µmol/mg prot) = y × Vsample ÷ (Vsample × Cpr) × n = y ÷ Cpr × nParameter Description:1 mM = 1 mmol/L;Vsample : Volume of sample added, 0.05 mL;n: Sample dilution factor;Cpr: Sample protein concentration, mg/mL;W: Sample weight, g;Vtotal: Total volume of sample extract, 1 mL;500: Cell or bacterial count, 5×10⁶, converted to units of 10⁴.Result Presentation Using Previous Standard CurveTypical Standard Curve: y = 2.2613x - 0.0531Example-1: 50 µL of chicken serum was taken and processed according to the assay steps using a 96-well plate. The measured ΔAtest = Atest - Ablank= 0.435 - 0.096 = 0.339. Substituting into the standard curve, y = 0.713 mM. Calculated based on liquid volume: Lactate content (mM) = y × n = 0.713 × 5 = 3.565 mM.PrecautionsIt is recommended to perform preliminary experiments using 2-3 samples expected to have significant differences before formal testing.For tissue and cell samples, results can be normalized by measuring the protein concentration.This kit is compatible with spectrophotometer detection. Adjust the preparation volume of detection reagents proportionally according to the spectrophotometer's requirements.It is recommended to establish your own standard curve for improved accuracy. If not, you may refer to the typical standard curve formula provided in the results section for calculation.Biochemical reagents are generally irritating and biologically toxic. For your safety and health, please wear appropriate personal protective equipment (lab coat, mask, gloves, hair cap, etc.) throughout the experiment and perform experiments in a fume hood or biosafety cabinet.This product is for scientific research use only. Not intended for clinical diagnosis.Frequently Asked QuestionsWhat should I do if the sample ΔAtest is too high or too low?If the sample ΔAtest is >1.0, the lactate content in the sample is too high. Dilute the sample appropriately with Lactate Assay Buffer (multiply by the dilution factor in the calculation). If the sample ΔAtest is <0.13, increase the sample amount... 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 | DescriptioniPE-Quick Kit is intended for the advanced confirmation of target protein expression utilizingE. Coliextract before the use of theiPE kit (Prod. No. 905089) | Product content: S665546Component50 TStorageS665546ABuffer QSL45 mLRTS665546BBuffer RIL11 mL2-8℃S665546CBuffer ML10 mLRTS665546DBuffer GW1 (concentrate)13 mLRTS665546EBuffer GW2 (concentrate)26 mLRTS665546FBuffer EBL13 mLRTS665546GRNase A240 µLRTS665546HLysis Tubes Ⅱ50 Product content: S665546Component50 TStorageS665546ABuffer QSL45 mLRTS665546BBuffer RIL11 mL2-8℃S665546CBuffer ML10 mLRTS665546DBuffer GW1 (concentrate)13 mLRTS665546EBuffer GW2 (concentrate)26 mLRTS665546FBuffer EBL13 mLRTS665546GRNase A240 µLRTS665546HLysis Tubes Ⅱ50 EARTS665546ISpin Columns DM With Collection Tubes50 EARTProduct IntroductionThis kit provides a method for extracting total DNA from soil or fecal samples, including the total DNA of cells, bacteria, parasites, and viruses in the samples. It is also suitable for extracting DNA from samples containing high concentrations of PCR reaction inhibitors. This reagent kit adopts a unique buffering system to efficiently bind DNA from the lysis solution to the adsorption column. Inhibitors of PCR and enzyme reactions, as well as residual impurities, can be effectively removed through 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 second-generation sequencing (16S amplicons and metagenomes), library construction, PCR, qPCR, Southern Blot, enzyme digestion molecular markers, etc.Self prepared reagents1. Constant temperature mixer - Product number: CW25932. Anhydrous ethanol, isopropanol3. Vortex oscillator or tissue grinderPreparation 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 (concentrate) and Buffer GW2 (concentrate) according to the instructions on the reagent bottle label.3. Take out the buffer RIL before use and store it at 2-8 ℃ immediately after use.Operation steps1. Centrifuge the Lysis Tube briefly to allow the beads to settle at the bottom.2. a. Add 0.1-0.3 g of soil or fecal sample to Lysis Tube, and add 740-820 µ L Buffer QSL and 4 µ L RNase A, tighten the tube cover and briefly vortex to mix.b. If fecal samples are stored in non lytic fecal preservation solutions (such as CWY041S and CWY041M), add 200 to Lysis Tube µ L-600 µ L solid-liquid mixture, centrifuge at 13000 rpm for 1 minute, discard the storage solution (if the amount of solid after centrifugation is too small, it can be enriched again, but should not exceed 0.3g). Join 620 µ LBuffer QSL and 4 µ L RNase A, tighten the tube cover and briefly vortex to mix.3. Fix the Lysis Tube in an oscillating grinding device equipped with a 2 mL adapter and process it according to the optimized grinding conditions of your equipment (see appendix).4. Shake the Lysis Tube on a constant temperature mixer at 70 ℃ and 1200 rpm for 10 minutes. Subsequently, centrifuge at 13000 rpm for 2 minutes to precipitate solid particles. Transfer 540 µ Transfer the supernatant to a new 2 mL centrifuge tube.5. Add 180 µ L Buffer RIL, vortex for 5 seconds, centrifuge at 13000 rpm for 2 minutes.Attention: Remove the buffer RIL before use and store it at 2-8 ℃ immediately after use.6. Add 160 to the new centrifuge tube in sequence µ L Buffer ML, 480 µ Supernatant from step 5, 320 µ L isopropanol, vortex for 5 seconds.7. Transfer the solution from the previous step to 650 µ Centrifuge at 12000 rpm (~13400 × g) for 1 minute into the spin columns DM that have been loaded into the collection tube.8. Discard the waste liquid in the collection pipe and place the adsorption column back into the collection pipe. Repeat step 7 until all the solution has been transferred.9. Add 500 to the adsorption column µ L 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.10. Add 500 to the adsorption column µ L 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. 11. Repeat step 10.12.12000 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.).13. Place the adsorption column in a new centrifuge tube (self provided) and add 50-200 drops of suspended droplets to the middle of the adsorption column µ L Buffer EBL 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) Incubating at room temperature for 5 minutes before centrifugation can increase yield.2) Use an additional 50-100 µ Further elution with L buffer or sterilized water can increase yield.3) If you want to increase the final concentration of DNA, you can add the DNA eluent obtained in step 13 back onto the adsorption membrane and repeat step 13, but it may reduce the total yield.4) The elution buffer does not contain chelating agents, please store DNA at -20 ℃.5) The residual trace PCR inhibitors in the genomic DNA template may have adverse effects on the PCR reaction, which can usually be resolved by diluting the DNA by 2-10 times.Appendix: Grind the sample using one of the following methods1. Manually vortex oscillate at maximum speed on the vortex oscillator for 10 minutes.2. On a vortex oscillator equipped with a 1.5-2 mL horizontal centrifuge tube holder, oscillate at maximum speed for 10 minutes (keeping the Lysis Tube horizontal). If the sample size exceeds 12, extend by 5-10 minutes. For example, using Scientific Industries or Mobile's Vortex Genie2 vortex oscillator.3.When using Qiagen's TissueLyser II, grind at 25Hz for 10 minutes.4.When using Qiagen's PowerLyzer 24 Homogenizer, homogenize at 2000 rpm for 30 seconds, pause for 30 seconds, and then homogenize again at 2000 rpm for 30 seconds.5.When using FastPrep-24 from MP Biomedicals, the recommended speed is 6.0 and the time is 40 seconds... Read More | Products contentProducts IntroductionThis kit is suitable for simple, rapid and efficient isolation and purification of DNA/RNA from whole blood, tissue homogenates, swabs, serum, plasma and other cell-free body fluids, etc. The unique buffer system enables the viral nucleic acids in the lysate to Products contentProducts IntroductionThis kit is suitable for simple, rapid and efficient isolation and purification of DNA/RNA from whole blood, tissue homogenates, swabs, serum, plasma and other cell-free body fluids, etc. The unique buffer system enables the viral nucleic acids in the lysate to bind to the silica gel centrifugal adsorbent columns in a highly efficient manner, and the viral nucleic acids obtained are of high purity and stable quality, free of protein, nuclease and other impurities, and can be used in a variety of routine operations, including PCR, fluorescence quantitative PCR and other experiments. It can be used for a variety of routine operations, including PCR, fluorescence quantitative PCR and other experiments.Bring your own instrumentsThermostatic mixer.Pre-experiment Preparation and Important Notes1. Read these instructions carefully before experimenting.2. If Proteinase K is to be stored for a long period of time, please keep it at -20℃.3. Check Buffer RLC for crystallization or precipitation prior to use, and if crystallization or precipitation occurs, redissolve Buffer RLC in a 56°C water bath.4. Pre-treatment of tissue samples: Take 20 mg of tissue samples into 1.5 mL centrifuge tubes (self-provided), add 500 µL of Buffer RLC, and after the tissue homogenizer breaks up, centrifuge the samples for 1 minute at 12,000 rpm (~13,400×g), and take 200 µL of supernatant as samples. procedure1. Take a 1.5 mL centrifuge tube (provided), add 500 µL of Buffer RLC, 200 µL of sample, 20 µL of Proteinase K, vortex for 5 s, and then place it in a thermostatic mixer at 1200 rpm for 10 min at room temperature. Note: For wet swab samples, 200 µL of sample was taken after sufficiently shaking and mixing. Note: For wet swabs, 200 µL was taken from the sample after it was soaked in 400 µL of saline, shaken and mixed thoroughly for 5 minutes, and then centrifuged at 12,000 rpm for 1 minute, and 200 µL was taken for extraction.2. Instantly remove the centrifuge tube and add the solution from step 1 to the Spin Columns DM in the collection tube. centrifuge at 12,000 rpm (~13,400 x g) for 1 minute, pour off the waste liquid from the collection tube, and return the column to the collection tube.3. Add 500 µL of Buffer PGWT to the adsorbent column, centrifuge at 12,000 rpm for 1 minute, pour off the waste liquid from the collection tube, and return the column to the collection tube.4. Add 500 µL of Buffer GWT2 to the adsorbent column, centrifuge at 12,000 rpm for 1 minute, pour off the waste liquid from the collection tube, and return the column to the collection tube.5. Centrifuge at 12,000 rpm for 2 minutes and pour off the waste liquid in the collection tube. Place the adsorption column at room temperature for 2 minutes and allow to dry.6. Place the column in a new collection tube (RNase-Free Centrifuge Tube), add 40-100 µL of RNase-Free Water to the center of the column membrane, let it stand at room temperature for 2 minutes, and then centrifuge at 12,000 rpm for 1 minute to collect the nucleic acid solution. Store at -80℃ for a long time... Read More |