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Human DNA (cytosine-5)-methyltransferase 1 (DNMT1) ELISA Kit (HUEB2173)

SKU:
HUEB2173
Product Type:
ELISA Kit
Size:
96 Assays
Uniprot:
P26358
Range:
0.156-10 ng/mL
ELISA Type:
Sandwich
Synonyms:
DNMT1, DNA, cytosine-5-methyltransferase 1
Reactivity:
Human
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Description

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Product Name:Human DNA (cytosine-5)-methyltransferase 1 (DNMT1) ELISA Kit
SKU:HUEB2173
Size:96T
Target:Human DNA (cytosine-5)-methyltransferase 1 (DNMT1)
Synonyms:CXXC-type zinc finger protein 9, DNA methyltransferase HsaI, MCMT, DNA MTase HsaI, Dnmt1, AIM, CXXC9, DNMT
Assay Type:Sandwich
Detection Method:ELISA
Reactivity:Human
Detection Range:0.156-10ng/mL
Sensitivity:0.078ng/mL
Intra CV:3.9%
Inter CV:6.2%
Linearity:
Sample1:21:41:81:16
Serum(N=5)83-92%102-112%103-116%96-105%
EDTA Plasma(N=5)104-114%109-118%102-112%107-116%
Heparin Plasma(N=5)108-108%108-117%95-107%85-94%
Recovery:
Sample TypeAverage(%)Recovery Range(%)
Serum10195-107
Plasma10397-109
Function:Methylates CpG residues. Preferentially methylates hemimethylated DNA. Associates with DNA replication sites in S phase maintaining the methylation pattern in the newly synthesized strand, that is essential for epigenetic inheritance. Associates with chromatin during G2 and M phases to maintain DNA methylation independently of replication. It is responsible for maintaining methylation patterns established in development. DNA methylation is coordinated with methylation of histones. Mediates transcriptional repression by direct binding to HDAC2. In association with DNMT3B and via the recruitment of CTCFL/BORIS, involved in activation of BAG1 gene expression by modulating dimethylation of promoter histone H3 at H3K4 and H3K9. Probably forms a corepressor complex required for activated KRAS-mediated promoter hypermethylation and transcriptional silencing of tumor suppressor genes (TSGs) or other tumor-related genes in colorectal cancer (CRC) cells (PubMed:24623306). Also required to maintain a transcriptionally repressive state of genes in undifferentiated embryonic stem cells (ESCs) (PubMed:24623306). Associates at promoter regions of tumor suppressor genes (TSGs) leading to their gene silencing (PubMed:24623306). Promotes tumor growth (PubMed:24623306).
Uniprot:P26358
Sample Type:Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:Natural and recombinant human DNA (cytosine-5)-methyltransferase 1
Sub Unit:Homodimer (PubMed:19173286). Forms a stable complex with E2F1, BB1 and HDAC1 (PubMed:10888886). Forms a complex with DMAP1 and HDAC2, with direct interaction (PubMed:10888872). Interacts with the PRC2/EED-EZH2 complex (PubMed:16357870). Probably part of a corepressor complex containing ZNF304, TRIM28, SETDB1 and DNMT1 (PubMed:24623306). Interacts with UHRF1; promoting its recruitment to hemimethylated DNA (PubMed:21745816). Interacts with USP7, promoting its deubiquitination (PubMed:21745816). Interacts with PCNA (PubMed:9302295). Interacts with MBD2 and MBD3 (PubMed:10947852). Interacts with DNMT3A and DNMT3B (PubMed:12145218). Interacts with UBC9 (PubMed:19450230). Interacts with CSNK1D (By similarity). Interacts with HDAC1 (By similarity). Interacts with BAZ2A/TIP5.
Research Area:Cancer
Subcellular Location:Nucleus
Storage:Please see kit components below for exact storage details
Note:For research use only
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:Methyltransferase, DNA; EC 2.1.1.37; Cell development/differentiation; Methyltransferase; Transcription regulation; Amino Acid Metabolism - cysteine and methionine

Chromosomal Location of Human Ortholog: 19p13.2

Cellular Component: nucleoplasm; centric heterochromatin; replication fork; nucleus

Molecular Function:DNA (cytosine-5-)-methyltransferase activity; protein binding; methyl-CpG binding; DNA binding; zinc ion binding; RNA binding; chromatin binding; DNA-methyltransferase activity; transcription factor binding

Biological Process: negative regulation of histone H3-K9 methylation; transcription, DNA-dependent; maintenance of DNA methylation; negative regulation of gene expression, epigenetic; DNA methylation; gene expression; negative regulation of transcription from RNA polymerase II promoter; positive regulation of histone H3-K4 methylation; chromatin modification; gene silencing; regulation of gene expression, epigenetic; DNA methylation on cytosine; regulation of cell proliferation

Disease: Cerebellar Ataxia, Deafness, And Narcolepsy, Autosomal Dominant; Neuropathy, Hereditary Sensory, Type Ie

NCBI Summary:DNA (cytosine-5-)-methyltransferase 1 has a role in the establishment and regulation of tissue-specific patterns of methylated cytosine residues. Aberrant methylation patterns are associated with certain human tumors and developmental abnormalities. Two transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Aug 2008]
UniProt Code:P26358
NCBI GenInfo Identifier:12231019
NCBI Gene ID:1786
NCBI Accession:P26358.2
UniProt Secondary Accession:P26358,Q9UHG5, Q9ULA2, Q9UMZ6, A0AV63, B7ZLW6,
UniProt Related Accession:P26358
Molecular Weight:1616
NCBI Full Name:DNA (cytosine-5)-methyltransferase 1
NCBI Synonym Full Names:DNA (cytosine-5-)-methyltransferase 1
NCBI Official Symbol:DNMT1
NCBI Official Synonym Symbols:AIM; DNMT; MCMT; CXXC9; HSN1E; ADCADN
NCBI Protein Information:DNA (cytosine-5)-methyltransferase 1; DNA (cytosine-5)-methyltransferase 1; m.HsaI; DNA MTase HsaI; DNA methyltransferase HsaI; CXXC-type zinc finger protein 9
UniProt Protein Name:DNA (cytosine-5)-methyltransferase 1
UniProt Synonym Protein Names:CXXC-type zinc finger protein 9; DNA methyltransferase HsaI; DNA MTase HsaI; M.HsaI; MCMT
UniProt Gene Name:DNMT1
UniProt Entry Name:DNMT1_HUMAN
Component Quantity (96 Assays) Storage
ELISA Microplate (Dismountable) 8×12 strips -20°C
Lyophilized Standard 2 -20°C
Sample Diluent 20ml -20°C
Assay Diluent A 10mL -20°C
Assay Diluent B 10mL -20°C
Detection Reagent A 120µL -20°C
Detection Reagent B 120µL -20°C
Wash Buffer 30mL 4°C
Substrate 10mL 4°C
Stop Solution 10mL 4°C
Plate Sealer 5 -

Other materials and equipment required:

  • Microplate reader with 450 nm wavelength filter
  • Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
  • Incubator
  • Deionized or distilled water
  • Absorbent paper
  • Buffer resevoir

*Note: The below protocol is a sample protocol. Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

Allow all reagents to reach room temperature (Please do not dissolve the reagents at 37°C directly). All the reagents should be mixed thoroughly by gently swirling before pipetting. Avoid foaming. Keep appropriate numbers of strips for 1 experiment and remove extra strips from microtiter plate. Removed strips should be resealed and stored at -20°C until the kits expiry date. Prepare all reagents, working standards and samples as directed in the previous sections. Please predict the concentration before assaying. If values for these are not within the range of the standard curve, users must determine the optimal sample dilutions for their experiments. We recommend running all samples in duplicate.

Step
1. Add Sample: Add 100µL of Standard, Blank, or Sample per well. The blank well is added with Sample diluent. Solutions are added to the bottom of micro ELISA plate well, avoid inside wall touching and foaming as possible. Mix it gently. Cover the plate with sealer we provided. Incubate for 120 minutes at 37°C.
2. Remove the liquid from each well, don't wash. Add 100µL of Detection Reagent A working solution to each well. Cover with the Plate sealer. Gently tap the plate to ensure thorough mixing. Incubate for 1 hour at 37°C. Note: if Detection Reagent A appears cloudy warm to room temperature until solution is uniform.
3. Aspirate each well and wash, repeating the process three times. Wash by filling each well with Wash Buffer (approximately 400µL) (a squirt bottle, multi-channel pipette,manifold dispenser or automated washer are needed). Complete removal of liquid at each step is essential. After the last wash, completely remove remaining Wash Buffer by aspirating or decanting. Invert the plate and pat it against thick clean absorbent paper.
4. Add 100µL of Detection Reagent B working solution to each well. Cover with the Plate sealer. Incubate for 60 minutes at 37°C.
5. Repeat the wash process for five times as conducted in step 3.
6. Add 90µL of Substrate Solution to each well. Cover with a new Plate sealer and incubate for 10-20 minutes at 37°C. Protect the plate from light. The reaction time can be shortened or extended according to the actual color change, but this should not exceed more than 30 minutes. When apparent gradient appears in standard wells, user should terminatethe reaction.
7. Add 50µL of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.
8. Determine the optical density (OD value) of each well at once, using a micro-plate reader set to 450 nm. User should open the micro-plate reader in advance, preheat the instrument, and set the testing parameters.
9. After experiment, store all reagents according to the specified storage temperature respectively until their expiry.

When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.

Sample Type Protocol
Serum If using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. If serum separator tubes are not being used, allow samples to clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.
Plasma Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples at 4°C for 15 mins at 1000 × g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. Note: Over haemolysed samples are not suitable for use with this kit.
Urine & Cerebrospinal Fluid Collect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid.
Cell culture supernatant Collect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately.
Cell lysates Solubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Tissue homogenates The preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenize in 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or -80°C.
Tissue lysates Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Breast Milk Collect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles.
ELISA
Human DNMT1 ELISA Kit

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