Rat DNMT1 (DNA Methyltransferase 1) ELISA Kit
This ELISA kit uses Sandwich-ELISA as the method. The micro ELISA plate provided in this kit has been pre-coated with an antibody specific to Rat DNMT1 . Standards or samples are added to the appropriate micro ELISA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for Rat DNMT1 and Avidin-Horseradish Peroxidase (HRP) conjugate are added to each micro plate well successively and incubated. Free components are washed away. The substrate solution is added to each well. Only those wells that contain Rat DNMT1, biotinylated detection antibody and Avidin-HRP conjugate will appear blue in color. The enzyme-substrate reaction is terminated by adding Stop Solution and the color turns yellow. The optical density (OD) is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The OD value is proportional to the concentration of Rat DNMT1. The concentration of Rat DNMT1 in samples can be calculated by comparing the OD of the samples to the standard curve.
|Detection Range||0.16-10 ng/mL|
|Sample Volume Required Per Well||100uL|
|Sample Type||Serum, plasma and other biological fluids|
This kit recognizes Rat DNMT1 in samples. No significant cross-reactivity or interference between Rat DNMT1 and analogues was observed.
As the OD values of the standard curve may vary according to the conditions of the actual assay performance (e.g. operator, pipetting technique, washing technique or temperature effects), the operator should establish a standard curve for each test. Typical standard curve and data is provided below for reference only.
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Rat DNMT1 were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Rat DNMT1 were tested on 3 different plates, 20 replicates in each plate.
|Intra-assay Precision||Inter-assay Precision|
|C V (%)||7.55||5.19||5.22||7.14||4.08||3.47|
The recovery of Rat DNMT1 spiked at three different levels in samples throughout the range of the assay was evaluated in various matrices.
|Sample Type||Range (%)||Average Recovery (%)|
|EDTA plasma (n=5)||85-97||91|
|Cell culture media (n=5)||90-103||95|
Samples were spiked with high concentrations of Rat DNMT1 and diluted with Reference Standard & Sample Diluent to produce samples with values within the range of the assay.
|Serum (n=5)||EDTA plasma (n=5)||Cell culture media (n=5)|
Kit Components & Storage
An unopened kit can be stored at 4'C for 1 month. If the kit is not used within 1 month, store the items separately according to the following conditions once the kit is received.
|Micro ELISA Plate(Dismountable)||8 wells X12 strips||-20'C, 6 months|
|Reference Standard||2 vials|
|Concentrated Biotinylated Detection Ab (100X)||1 vial, 120 uL|
|Concentrated HRP Conjugate (100X)||1 vial, 120 uL||-20'C(shading light), 6 months|
|Reference Standard & Sample Diluent||1 vial, 20 mL||4'C, 6 months|
|Biotinylated Detection Ab Diluent||1 vial, 14 mL|
|HRP Conjugate Diluent||1 vial, 14 mL|
|Concentrated Wash Buffer (25X)||1 vial, 30 mL|
|Substrate Reagent||1 vial, 10 mL||4'C(shading light)|
|Stop Solution||1 vial, 10 mL||4'C|
|Plate Sealer||5 pieces|
|Product Description||1 copy|
|Certificate of Analysis||1 copy|
Rat DNMT1 (DNA Methyltransferase 1) ELISA Kit (RTES00907) Assay procedure
- 1. Set standard, test sample and control (zero) wells on the pre-coated plate and record theirpositions. It is recommended to measure each standard and sample in duplicate. Note: addall solutions to the bottom of the plate wells while avoiding contact with the well walls. Ensuresolutions do not foam when adding to the wells.
- 2. Aliquot 100µl of standard solutions into the standard wells.
- 3. Add 100µl of Sample / Standard dilution buffer into the control (zero) well.
- 4. Add 100µl of properly diluted sample (serum, plasma, tissue homogenates and otherbiological fluids) into test sample wells.
- 5. Cover the plate with the sealer provided in the kit and incubate for 90 min at 37°C.
- 6. Aspirate the liquid from each well, do not wash. Immediately add 100µL of BiotinylatedDetection Ab working solution to each well. Cover the plate with a plate seal and gently mix.Incubate for 1 hour at 37°C.
- 7. Aspirate or decant the solution from the plate and add 350µL of wash buffer to each welland incubate for 1-2 minutes at room temperature. Aspirate the solution from each well andclap the plate on absorbent filter paper to dry. Repeat this process 3 times. Note: a microplatewasher can be used in this step and other wash steps.
- 8. Add 100µL of HRP Conjugate working solution to each well. Cover with a plate seal andincubate for 30 min at 37°C.
- 9. Aspirate or decant the solution from each well. Repeat the wash process for five times asconducted in step 7.
- 10. Add 90µL of Substrate Reagent to each well. Cover with a new plate seal and incubate forapproximately 15 min at 37°C. Protect the plate from light. Note: the reaction time can beshortened or extended according to the actual color change, but not by more than 30min.
- 11. Add 50 µL of Stop Solution to each well. Note: Adding the stop solution should be done inthe same order as the substrate solution.
- 12. Determine the optical density (OD value) of each well immediately with a microplate readerset at 450 nm.
Rat DNMT1 (DNA Methyltransferase 1) ELISA Kit (RTES00907) Protein Information
|UniProt Protein Function:||DNMT1: an ubiquitous DNA methyltransferase that methylates CpG residues. Preferentially methylates hemimethylated DNA. It is responsible for maintaining methylation patterns established in development, and may play an active role in DNA damage repair. Mediates transcriptional repression by direct binding to HDAC2. Its abundance is reduced to non detectable levels at the G0 phase of the cell cycle and is dramatically induced upon entrance into the S-phase of the cell cycle. Interacts with HDAC1 and with PCNA. Forms a complex with DMAP1 and HDAC2, with direct interaction. Forms also a stable complex with E2F1, BB1 and HDAC1. Binds MBD2 and MBD3. Three isoforms of the human protein produced by alternative splicing have been described.|
|UniProt Protein Details:|
Protein type:Amino Acid Metabolism - cysteine and methionine; Cell development/differentiation; EC 18.104.22.168; Methyltransferase; Methyltransferase, DNA; Transcription regulation
Chromosomal Location of Human Ortholog: 8q13
Cellular Component: cell soma; centric heterochromatin; cytoplasm; heterochromatin; nucleus; protein complex; replication fork
Molecular Function:chromatin binding; DNA (cytosine-5-)-methyltransferase activity; DNA binding; DNA-methyltransferase activity; estrogen receptor binding; histone deacetylase binding; methyl-CpG binding; methyltransferase activity; protein binding; protein domain specific binding; RNA binding; S-adenosylmethionine-dependent methyltransferase activity; zinc ion binding
Biological Process: aging; brain development; covalent chromatin modification; cytosine methylation within a CG sequence; DNA methylation; DNA methylation involved in embryo development; DNA methylation on cytosine; gene silencing; maintenance of DNA methylation; negative regulation of histone H3-K9 methylation; negative regulation of transcription from RNA polymerase II promoter; negative regulation of transcription, DNA-dependent; neuron differentiation; positive regulation of histone H3-K4 methylation; Ras protein signal transduction; regulation of cell proliferation; regulation of gene expression; response to activity; response to caffeine; response to drug; response to estradiol; response to ethanol; response to heat; response to ionizing radiation; response to lead ion; response to lipopolysaccharide; response to nutrient levels; response to organic substance; response to testosterone stimulus; response to toxin; response to vitamin A; S-adenosylmethionine metabolic process; transcription, DNA-dependent
|NCBI Summary:||methylates genomic DNA; involved in processes of gene inactivation, chromatin organization, X chromosome inactivation and genomic imprinting [RGD, Feb 2006]|
|NCBI GenInfo Identifier:||20137608|
|NCBI Gene ID:||84350|
|UniProt Related Accession:||Q9Z330|
|NCBI Full Name:||DNA (cytosine-5)-methyltransferase 1|
|NCBI Synonym Full Names:||DNA methyltransferase 1|
|NCBI Official Symbol:||Dnmt1|
|NCBI Protein Information:||DNA (cytosine-5)-methyltransferase 1|
|UniProt Protein Name:||DNA (cytosine-5)-methyltransferase 1|
|UniProt Synonym Protein Names:||DNA MTase RnoIP; M.RnoIP; DNA methyltransferase I; MCMT|
|UniProt Gene Name:||Dnmt1|