| Description | ATP is the most fundamental energy currency in living organisms, and its concentration directly affects the energy metabolism of various organs. As the most important energy molecule, ATP plays a critical role in diverse physiological and pathological processes. Changes in ATP levels can ATP is the most fundamental energy currency in living organisms, and its concentration directly affects the energy metabolism of various organs. As the most important energy molecule, ATP plays a critical role in diverse physiological and pathological processes. Changes in ATP levels can impact numerous cellular functions. Typically, ATP levels decrease during apoptosis, necrosis, or under certain toxic conditions, while high glucose stimulation can upregulate intracellular ATP levels in some cells. A decrease in ATP levels often indicates impaired or declining mitochondrial function. During apoptosis, the drop in ATP levels usually occurs simultaneously with a decrease in mitochondrial membrane potential. The ATP Assay Kit can be used to detect ATP levels in common solutions, cells, or tissues. This kit is developed based on the principle that firefly luciferase requires ATP to provide energy for catalyzing the production of light from luciferin. When both firefly luciferase and luciferin are in excess, the light produced is proportional to the ATP concentration within a certain range. This allows for highly sensitive detection of ATP concentration in solutions.E1501756Component200TStorageE1501756AATP Detection Reagent25 mL-20℃. Store in the dark.E1501756BATP Standard Solution100 µL-20℃. Store in the dark.E1501756CATP Assay Lysis Buffer100 mL-20℃. Store in the dark.Product Advantages1. High Sensitivity: Provides excellent detection results within the range of 0.1 nM to 100 µM.2. High Stability: ATP measurement results from prepared samples decrease by no more than 10% within 30 minutes.3. Good Compatibility of Prepared Samples: Cell or tissue samples lysed using the ATP Assay Lysis Buffer provided in this kit can not only be used for ATP detection but also for protein concentration assays, SDS-PAGE, or Western blotting for some commonly soluble proteins.4. Convenient and Fast: Typically, 10-20 samples can be assayed within 30-60 minutes.5. Simple Sample Preparation: Samples do not require perchloric acid or trichloroacetic acid (TCA) extraction. The specialized lysis buffer provided allows samples to be used for ATP detection after simple lysis.Experimental Procedure1. Sample PreparationNote: Sample lysis should be performed at 4°C or on ice.1.1 For Adherent CellsRemove the culture medium. Add Lysis Buffer according to the proportion of 200 µL per well of a 6-well plate (i.e., 1/10 of the 2 mL culture medium volume) to lyse the cells. For complete lysis, pipette up and down repeatedly or shake the plate to ensure the lysis buffer fully contacts and lyses the cells. Cells typically lyse immediately upon contact with the buffer. Centrifuge the lysate at 10,000 rpm, 4°C for 5 minutes. Collect the supernatant for subsequent assay.1.2 For Suspension CellsCentrifuge to pellet the cells, discard the supernatant, and gently resuspend the pellet. Add Lysis Buffer according to the proportion of 200 µL per the cell amount from one well of a 6-well plate. For complete lysis, tap the tube bottom or vortex appropriately to ensure the lysis buffer fully contacts and lyses the cells. Cells typically lyse immediately. Centrifuge the lysate at 10,000 rpm, 4°C for 5 minutes. Collect the supernatant for subsequent assay.1.3 For Tissue SamplesAdd Lysis Buffer in a ratio of approximately 100-200 µL per 20 mg of tissue. Homogenize using a glass homogenizer or other homogenization equipment. Thorough homogenization ensures complete tissue lysis. Centrifuge the lysate at 10,000 rpm, 4°C for 5 minutes. Collect the supernatant for subsequent assay.2. Standard Curve PreparationThaw the required reagents on ice. Dilute the ATP Standard Solution with ATP Assay Lysis Buffer to create appropriate concentration gradients. The specific concentrations should be determined based on the expected ATP concentration in the samples. For initial detection, concentrations of 0.01, 0.03, 0.1, 0.3, 1, 3, and 10 µM can be tested. In subsequent experiments, adjust the standard concentration range appropriately based on sample ATP levels.TubeLysis Buffer Volume (µL)ATP Standard Solution VolumeFinal Concentration (µM)A982 µL from stock (0.5 mM)10B7030 µL from Tube A3C9010 µL from Tube A1D9010 µL from Tube B0.3E9010 µL from Tube C0.1F9010 µL from Tube D0.03G9010 µL from Tube E0.013. ATP Concentration Measurement3.1 Add 100 µL of ATP Detection Reagent to each assay well. Incubate at room temperature for 3-5 minutes.3.2 Add 10 µL of sample or the diluted ATP standard solution to the assay well.3.3 Measure the Relative Light Unit (RLU) value using a luminometer.Note: The sample volume can be adjusted within the range of 10-100 µL. If the ATP concentration in the sample is low, 100 µL can be added. If the ATP concentration is high, a smaller volume can be used, but the same volume must be used for the standard curve samples. If the ATP concentration is exceptionally high, dilute the sample with ATP Assay Lysis Buffer before measurement.Precautions1. The Detection Reagent contains luciferase. Repeated freeze-thaw cycles will lead to gradual inactivation. For optimal performance, consider aliquoting after the first thaw, ensuring the aliquot containers are free from ATP contamination.2. Luciferase activity is temperature-sensitive. Before the reaction, equilibrate both cells and the ATP Detection Reagent to room temperature for measurement. Do not store at room temperature for extended periods.3. ATP, especially in lysed samples, is unstable at room temperature. Perform operations at 4°C or on ice.4. Use white or black 96-well or 384-well plates suitable for cell culture for detection. Using standard transparent plates may cause interference between adjacent wells.5. The provided ATP Assay Lysis Buffer effectively lyses and releases ATP from common cultured cells and tissues. For special tissues or samples where detected ATP levels are significantly lower than expected, boil a portion of the lysate for 2 minutes before centrifugation to fully release ATP. Boiling will denature proteins, which will precipitate during subsequent centrifugation; therefore, boiled samples cannot be used for protein concentration assays, SDS-PAGE, or Western blotting. Use the remaining portion of the sample for protein assays, SDS-PAGE, and Western blotting.6. For your safety and health, wear a lab coat and disposable gloves during operation... Read More | 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 | This reagent kit uses highly sensitive silver dye, which can be applied to protein staining of denatured and non denatured gels. It has the advantages of clear target bands, low background, and flexible control of operation time. In addition, this reagent kit has added a short-term sensitization This reagent kit uses highly sensitive silver dye, which can be applied to protein staining of denatured and non denatured gels. It has the advantages of clear target bands, low background, and flexible control of operation time. In addition, this reagent kit has added a short-term sensitization step, which can significantly reduce the background and enhance the brightness of the target band. P665901Component20 TStorageP665901ASilver Stain Sensitizer (500×)2×1 mLRTP665901BSilver Stain Enhancer3 mLRTP665901CSilver Stain2×250 mLRTP665901DSilver Stain Developer4×125 mLRT Matters needing attention1. Please prepare 50 ml of fixed solution (ultrapure water: ethanol: acetic acid=6:3:1), 50 ml of eluent (10% ethanol), and 50 ml of termination solution (5% acetic acid) in advance.2. Please use deionized water and clean glass or plastic containers during operation, and wear disposable gloves for operation.The entire silver dyeing process needs to be carried out on a shaker, with a rotation speed of about 60 rpm.4. Self prepared ethanol and glacial acetic acid are required.Instructions for useThe dosage of each solution in the following operation steps takes the gel with a size of 8.5 × 5.5 cm and a thickness of 1.0 mm as an example. The gel is immersed in the solution completely, and is operated on a shaker, with a general dosage of 25 ml. For large gel, the dosage of each solution should be scaled up according to the gel volume. Please prepare 50 ml of fixed solution (ultrapure water: ethanol: glacial acetic acid=6:3:1), 50 ml of eluent (10% ethanol), and 50 ml of termination solution (5% glacial acetic acid) in advance.1. Water washing: After electrophoresis is completed, wash the gel twice with ultrapure water for 5 minutes each time.2. Fixation: Fix the gel twice with 25 ml of fixative solution for 15 minutes each time.3. Elution: Wash the adhesive twice with eluent, each time for 5 minutes.4. Water washing: Wash the glue twice with ultrapure water, each time for 5 minutes.5. Sensitization: put the gel washed in the previous step into the silver dye sensitization working solution, incubate it accurately for 1 minute at room temperature, and then wash it with ultrapure water for three times, each time for 20 seconds. Preparation of silver staining sensitization working solution: Take 50 µ l Silver Stain Sensitivity (500 x) and add it to 25 ml of ultrapure water, mix well.6. Silver staining: discard ultrapure water and incubate gel in silver staining working solution for 30 minutes. Preparation of silver staining working solution: Take 25ml Silver Stain and add 50 µ l Silver Stain Enhanced to mix well.7. Water washing: Quickly wash the glue twice with ultrapure water, with each washing accurately controlled for 20 seconds.8. Development: Immerse the washed gel in the developer immediately and incubate it at room temperature for 2-3 minutes until the protein strip is clear. Preparation of developer: Take 25ml Silver Stain Developer and add 30 µ l Silver Stain Enhanced to mix well. Attention: Within 30 seconds of development, protein bands begin to appear and continue to develop for 2-3 minutes. If the protein band appears lighter, the development time can be appropriately extended to 5 minutes or more.9. Termination: After washing the developer on the gel with the termination solution, soak the gel in a new termination solution to react for 10 minutes.Experimental imagesSilver staining results of BSA protein samples after 10% SDS-PAGE gel electrophoresisThe molecular weight of BSA protein is about 66 kD, and the loading amounts from left to right are 50 ng, 10 ng, and 5 ng, respectively... Read More | DescriptionThe Universal Coupling Kit makes particle-based immunoassays, lateral flow tests and biomolecule separation applications more flexible than ever before. It is the only kit that allows users to select and couple their choice of carboxylated particle with their chosen protein.Employing a DescriptionThe Universal Coupling Kit makes particle-based immunoassays, lateral flow tests and biomolecule separation applications more flexible than ever before. It is the only kit that allows users to select and couple their choice of carboxylated particle with their chosen protein.Employing a unique mechanism to immobilise proteins, Anteo′s advantages outweigh those of conventional covalent chemistries such as NHS/EDC or passive binding. This facilitates coupling of antibodies with ease, improved functionality and reproducibility, leading to better uniformity between experiments.Anteo′s Activation Reagent is water-based and replaces the dry chemicals you would use with the traditional NHS/EDC method. Our One-Step-Activation only takes one hour, and improves efficiency in terms of both time and cost. It also provides the ability to either store activated particles up to 12 months for later use, or to immediately couple proteins.Particle-Based Immunoassays, Lateral Flow, Bioseparations and Immunoprecipitation... Read More | V669947 Component 50T Storage V669947A Buffer GL 15 mL RT V669947B Buffer GW1 (concentrate) 13 mL RT V669947C Buffer GW2 (concentrate) 15 mL RT V669947D Buffer RE 10 mL RT V669947E Proteinase K 12.5 mg RT V669947F Proteinase K Storage Buffer 1.25 mL RT V669947G Spin Columns RS with Collection Tubes V669947 Component 50T Storage V669947A Buffer GL 15 mL RT V669947B Buffer GW1 (concentrate) 13 mL RT V669947C Buffer GW2 (concentrate) 15 mL RT V669947D Buffer RE 10 mL RT V669947E Proteinase K 12.5 mg RT V669947F Proteinase K Storage Buffer 1.25 mL RT V669947G Spin Columns RS with Collection Tubes 50 RT V669947H RNase-Free Centrifuge Tubes (1.5 mL) 50 RTProductsThis kit is suitable for the extraction of viral RNA and DNA from fresh or frozen plasma, serum and cell-free body fluids. It is easy to operate as it does not require the use of organic solvents such as phenol and chloroform for extraction. The kit uses a unique buffer system to enable efficient and specific binding of viral nucleic acids in lysate to silica gel centrifugal adsorption columns. Inhibitors of PCR and enzyme reactions as well as residual impurities can be efficiently removed in a two-step effective rinsing step, and finally high purity viral nucleic acids can be obtained by using a low-salt buffer or water for elution. The purified viral nucleic acid is free of protein, nuclease and other impurities, and can be used directly in PCR, RT-PCR, Real-Time PCR, blotting experiments and so on.Self-contained reagent: anhydrous ethanol.Pre-experiment and Important Notes1. Add 1.25ml 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, and avoid repeated freezing and thawing to avoid affecting its activity. Do not add Proteinase K directly into Buffer GL.2. Repeated freezing and thawing of the sample should be avoided, as this may result in smaller DNA fragments and a decrease in the amount of extracted DNA.3. Avoid repeated freezing and thawing of serum or plasma, which can lead to protein denaturation or precipitation, reducing the viral titer and thus affecting the yield of extracted viral nucleic acids.4. Anhydrous ethanol should be added to Buffer GW1 and Buffer GW2 according to the label instructions of the reagent bottle before first use.5. Check Buffer GL for crystallization or precipitation before use. If crystallization or precipitation occurs, redissolve Buffer GL in a water bath at 56℃.Procedure1. Take a 1.5 ml centrifuge tube (self-provided) and add 20 µl Proteinase K.2. Add 200 µl serum or plasma to the centrifuge tube. Add 200µl Buffer GL and vortex and shake for 15 seconds.Note: 1) Sample volume less than 200 µl can be made up by adding 0.9% NaCl (self-provided). 2) In order to ensure effective lysis of the sample, the sample needs to be mixed well with Buffer GL after adding Buffer GL.3. Incubate at 56°C for 15 minutes, centrifuge briefly, and collect the solution from the wall of the tube to the bottom of the tube.4. 250 µl of anhydrous ethanol was added, vortexed and shaken for 15 seconds, left at room temperature for 5 minutes, centrifuged briefly, and the solution on the wall of the tube was collected at the bottom of the tube.Note: If the ambient temperature exceeds 25°C, anhydrous ethanol should be used after pre-cooling on ice.5. Add the solution obtained in step 4 to the adsorbent column (RNase-Free Columns RS) that has been loaded into the collection tube, and if the solution cannot be added at one time, it can be transferred in several times. centrifuge the column at 12,000 rpm (~13,400 × g) for 1 min, pour off the waste liquid in the collection tube, and put the column back into the collection tube.6. Add 500 µl of Buffer GW1 to the adsorption column (check that anhydrous ethanol has been added before use), centrifuge at 12,000 rpm for 1 minute, pour off the waste liquid in the collection tube, and put the adsorption column back into the collection tube.7. Add 500 µl of Buffer GW2 to the adsorption column (check that anhydrous ethanol has been added before use), centrifuge at 12,000 rpm for 1 minute, pour off the waste liquid in the collection tube, and put the adsorption column back into the collection tube.Note: Step 7 can be repeated if further DNA purity is required.8. Add 500 µl of anhydrous ethanol to the adsorbent column and centrifuge at 12,000 rpm for 1 min. Pour off the waste liquid in the collection tube and put the adsorbent column back into the collection tube.9. Centrifuge at 12,000 rpm for 3 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 the removal of residual ethanol from the adsorbent column; ethanol residue can interfere with subsequent enzymatic reactions (digestion, PCR, etc.).10. Place the adsorption column in a new collection tube (RNase-Free Centrifuge Tube), add 20-150 µl of Buffer RE or sterilized water overhanging the middle of the adsorption column membrane, leave it at room temperature for 2-5 minutes, and then centrifuge it at 12,000 rpm for 1 minute to collect the nucleic acid solution.Note: 1) If the downstream experiment is sensitive to pH or EDTA, you can use sterilized water for elution. The pH of the eluent has a great influence on the elution efficiency, if water is used as the eluent it should be ensured that its pH is 7.0-8.5 (the pH of water can be adjusted to this range with NaOH), and the elution efficiency is not high when the pH is lower than 7.0.(2) For long-term storage, please store the DNA solution at -20℃ and the RNA solution at -70℃.3) If the final concentration of DNA/RNA is to be increased, the DNA/RNA eluate obtained in step 10 can be re-spiked onto the adsorbent membrane and step 10 repeated... Read More |