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Description
Human DNMT1 ELISA KitProduct Specification Usage Self prepared test equipment required for the experiment: 1. plate reader ( 450nm 2. high precision sampler and gun head: 0. 5 10uL, 5 50uL, 20 200uL, 200 1000uL 3. 37 Incubator 4. Distilled water or deionized water, Sample handling and requirements: Tissue homogenate: with pre cooled PBS 0. 01M,pH=7. 4 ) Rinse the tissue to remove residual blood (lysed red blood cells in the homogenate will affect the measurement result),
Product Specification
| Usage |
Self-prepared test equipment required for the experiment: 1. plate reader ( 450nm ） 2. high-precision sampler and gun head: 0.5-10uL, 5-50uL, 20-200uL, 200-1000uL 3. 37℃℃ Incubator 4. Distilled water or deionized water, Sample handling and requirements: Tissue homogenate: with pre-cooled PBS （ 0.01M,pH=7.4 ) Rinse the tissue to remove residual blood (lysed red blood cells in the homogenate will affect the measurement result), and break the tissue after weighing. Combine the fragmented tissue with the corresponding volume PBS (Generally according to 1 ： 9 Weight to volume ratio, such as 1g The tissue samples correspond to 9mL Of PBS The specific volume can be appropriately adjusted according to the experimental needs and recorded. Recommended in PBS Add protease inhibitor) into a glass homogenizer and ground thoroughly on ice. For further lysis of tissue cells, the homogenate can be sonicated, or freeze-thawed repeatedly. Finally, the homogenate was mixed in 5000×g Centrifugation 5~10 Minutes, take the supernatant for detection. Cell lysate: pre-cooling for adherent cells PBS Gently washed, followed by trypsinization, 1000×g Centrifugation 5 Cells were collected after minutes; The suspended cells can be collected directly by centrifugation. The collected cells were pre-cooled with PBS Washing 3 Times, every 1×10^6 Added to cells 150-200uL PBS Resuspension (recommended at PBS Adding a protease inhibitor; If the content is very low, it can be appropriately reduced PBS Volume) and the cells were disrupted by repeated freeze-thaw or sonication. The extracts were mixed in 2-8℃ ， 1500×g Centrifugation 10 Minutes, take the supernatant for detection. Other biological fluids: 1000xg Centrifugation 20 Minutes, take the supernatant to detect. Preparations before testing: 1 , please advance 10 Minutes remove the kit from the refrigerator and equilibrate to room temperature. 2 , Standard gradient working solution preparation: add 1mL Universal diluent into lyophilized standard and let stand 15 Minutes until it is completely dissolved and then gently mix (the concentration is 10ng/mL ） And then according to the following concentrations: 10ng/mL 、 5ng/mL 、 2.5ng/mL 、 1.25ng/mL 、 0.625ng/mL 、 0.3125ng/mL 、 0.15625ng/mL 、 0ng/mL The dilution was performed. Double dilution method : Take 7 branch EP Tube , added to each tube 500uL Universal diluent ,10ng/mL Pipette from the standard working solution 500uL To the first EP Mix evenly in a tube 5ng/mL Standard Working Solution , according to this step, absorb and mix evenly in turn. The last tube is directly used as a blank hole , there is no need to suck liquid from the penultimate tube, as shown in the figure below. 3 Preparation of biotinylated antibody detection working solution: before use 15 Min. The concentrated biotinylated antibody was concentrated in 1000×g Centrifugation 1 Minutes, with a universal diluent 100× The concentrated biotinylated antibody was diluted into 1× Working concentration (ex: 10uL Concentrate +990uL Universal Diluent) , now available for use. 4 Preparation of enzyme conjugate working solution: before use 15 Minutes will 100× Concentrated enzyme conjugate in 1000×g Centrifugation 1 Minutes, with a universal diluent 100× concentrate HRP The enzyme conjugate is diluted into 1× Working concentration (ex: 10uL Concentrate +990uL Universal Diluent) , now available for use. 5 、 1× Wash liquid preparation: Take 10mL 20× Wash liquid to 190mL In distilled water (the concentrated washing liquid taken out of the refrigerator may have crystals, which is a normal phenomenon. It can be left at room temperature and prepared after the crystals are completely dissolved). Operation steps: 1 Equilibration from room temperature 10 After minutes, remove the required slats from the aluminum foil bag, and seal the remaining slats with ziplock bag and put them back 4℃ 。 2 , Adding samples: respectively add samples or different concentration standards according to 100ul Each well is added to the corresponding well, and the blank well is added 100uL Universal diluent. After covering the sealing film 37℃ Incubation 60 Minutes. (Recommendation : Minimum dilution of sample to be tested with universal diluent 1 Times later, add the enzyme labeled plate for testing. So as to reduce the influence of matrix effect on the test results, and finally, the sample concentration needs to be multiplied by the corresponding dilution factor when calculating. It is recommended to set up double wells for all samples and standards to be tested during testing). 3 Add biotinylated antibody: take out the enzyme plate, discard the liquid without washing. Directly add biotinylated antibody working solution to each well 100uL , after covering the sealing film 37℃ Incubation 60 Minutes. 4 Plate washing: discard the liquid and add to each well 300uL 1x Wash liquid, stand 1 Minutes, throw off the washing liquid, pat dry on absorbent paper, and repeat washing the plate 3 Times (the plate can also be washed with a plate washing machine). 5 Adding enzyme conjugate working solution: adding enzyme conjugate working solution to each well 100uL , after covering the sealing film 37℃ Incubation 30 Minutes. 6 Plate washing: discard the liquid according to the steps 4 Washing method, wash plate 5 Times. 7 Substrate addition: substrate is added per well ( TMB ） 90uL Covered with a sealing film, 37℃ Incubation protected from light 15 Minutes. 8 Add stop solution: take out the enzyme label plate and directly add stop solution to each well 50uL , immediately in 450nm Wavelength measurement of each well OD Value. Calculation of experimental results: Result judgment: 1 , calculate the average of the standard and sample replica well OD Value and subtract the blank hole's OD Values as correction values. Taking concentration as the abscissa, OD Value is ordinate , draw the standard curve of the four-parameter logic function on the double logarithmic coordinate paper. 2 If the sample OD If the value is higher than the upper limit of the standard curve, the test should be retested after appropriate dilution and multiplied by the corresponding dilution factor when calculating the sample concentration. |
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| Theory | This kit uses double antibody sandwich enzyme-linked immunosorbent assay (ELISA). To the microwells pre-coated with DNA Methyltransferase 1 (DNMT1) capture antibody, sample, standard product, biotin-labeled detection antibody, and HRP enzyme conjugate were added in sequence, incubated and washed in the middle, and colored with substrate TMB. TMB is converted to blue under the catalysis of peroxidase (HRP) and to final yellow under the action of acid. There was a positive correlation between the depth of color and DNA Methyltransferase 1 (DNMT1) in the sample. The absorbance (0D value) was measured with a microplate reader at a wavelength of 450nm, and the sample concentration was calculated. | |||||||||||||||||||||||||||||||||
| Source | Human | |||||||||||||||||||||||||||||||||
| Synonym | Human DNA Methyltransferase 1 ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | DNA methyltransferase 1 (DNMT1) is an enzyme that catalyzes the transfer of methyl groups to specific CpG structures in DNA, a process called DNA methylation. It is encoded by the DNMT1 gene. It is part of the DNA methyltransferase family, which consists primarily of DNMT1, DNMT3A, and DNMT3B. The enzyme is responsible for maintaining DNA methylation, thereby ensuring the fidelity of this epigenetic pattern in cell division. However, it can catalyze novel DNA methylation in specific genomic settings, including transposable elements and paternal imprinted control regions. | |||||||||||||||||||||||||||||||||
| General Notes | 1. Carry out incubation in strict accordance with the specified time and temperature to ensure accurate results. All reagents must reach room temperature 20-25 °C prior to use. Store reagents in refrigeration immediately after use. 2. Incorrect plate washing may lead to inaccurate results. Make sure to drain the liquid from the wells as much as possible before adding the substrate. Do not allow the wells to dry out during incubation. 3. Eliminate the residual liquid and fingerprints at the bottom of the plate, otherwise it will affect the OD value. 4. The substrate color development solution should be colorless or very light in color, and the substrate solution that has turned blue cannot be used. 5. Avoid cross-contamination of reagents and specimens to avoid wrong results. 6. Avoid direct exposure to strong light during storage and incubation. 7. Any reaction reagent cannot come into contact with the bleaching solvent or the strong gas emitted by the bleaching solvent. Any bleaching component will destroy the biological activity of the reaction reagents in the kit. 8. Expired products cannot be used, and components with different item numbers and batch numbers cannot be mixed. 9. Recombinant proteins from sources other than the kit may not match the antibodies in this kit and are not recognized. 10. If the disease may be spread, all samples should be managed well, and the samples and testing devices should be handled according to the prescribed procedures. |
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| Storage Temp. | Store the unopened kit at 4 °C. Shelf life: 6 months. | |||||||||||||||||||||||||||||||||
| Test Range | 0.156-10ng/mL | |||||||||||||||||||||||||||||||||
| Applications | Tissue homogenates, cell lysates and other biological fluids |
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