Dnmt1-Trap A

ABIN1082205 产品详细信息, 供应商: Log in to see
抗原
  • cb1075
  • cb983
  • dmnt1
  • fb05h01
  • mta
  • wu:fb05h01
  • ADCADN
  • AIM
  • CXXC9
  • DNMT
  • HSN1E
  • MCMT
  • DDM2
  • DECREASED DNA METHYLATION 2
  • DMT01
  • DMT1
  • DNA METHYLTRANSFERASE
  • DNA METHYLTRANSFERASE 01
  • DNA METHYLTRANSFERASE 1
  • MET2
  • METHYLTRANSFERASE 2
  • METHYLTRANSFERASE I
  • METI
  • methyltransferase 1
  • Cxxc9
  • Dnmt
  • Dnmt1o
  • MTase
  • Met-1
  • Met1
  • MommeD2
  • m.MmuI
  • DNA (cytosine-5-)-methyltransferase 1
  • DNA (cytosine-5)-methyltransferase 1
  • DNA methyltransferase (cytosine-5) 1
  • DNMT1
  • dnmt1
  • Dnmt1
  • MET1
适用

14
6
5
1
1
宿主
Camelidae
抗体类型
Recombinant Antibody
标记
Agarose Beads
应用范围
Affinity Measurement (AM), Chromatin Immunoprecipitation (ChIP), Enzyme Activity Assay (EAA), Immunoprecipitation (IP), Mass Spectrometry (MS), Protein Complex Immunoprecipitation (Co-IP), Pull-Down Assay (Pull-Down), Purification (Purif)
选项
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原理 Dnmt1-Trap is a high quality Dnmt1-binding protein coupled to a monovalent matrix (agarose particles) for biochemical analysis of Dnmt1 and interacting partners.
样品类型 Cell Extracts
特异性 Binding capacity: 10 µL Dnmt1-Trap_A slurry binds 2.5 - 3 µg of DNMT1
产品特性 Antibodies - extremely powerful tools in biomedical research - are large complex molecules (~ 150 kDa) consisting of two heavy and two light chains. Due to their complex structure, the use of antibodies is often limited and hindered by batch-to-batch variations.

Camelidae (camels, dromedaries, llamas and alpacas) possess functional antibodies devoid of light chains, so-called heavy chain antibodies (hcAbs). hcAbs recognize and bind their antigens via a single variable domain (VHH). These VHH domains are the smallest intact antigen binding fragments (~ 13 kDa).

Nano-Traps are based on single domain antibody fragments (VHHs) derived from alpaca.
组件 Dnmt1-Trap coupled to agarose beads
试剂未包括 Lysis buffer (CoIP), 10x RIPA buffer, Dilution buffer, Wash buffer, Elution buffer
别名 Dnmt1 (DNMT1 ELISA Kit 摘要)
背景 DNA methylation is the postreplicative transfer of a methyl group to the C5 position of cytosine bases. It was the first epigenetic modification identified and has been intensively studied for more than half a century. By now it is clear that Dnmt1, the major eucaryotic DNA methyltransferase, faithfully maintains genome-wide methylation patterns and plays an essential role in the epigenetic network controlling gene expression and genome stability during development. However, the molecular mechanisms that ultimately control DNA methylation still remain elusive.
研究领域 Chromatin and Nuclear Signaling
应用备注 For biochemical analyses including mass spectroscopy and enzyme activity measurements, DNMT1 and interacting factors can be isolated fast and efficiently (one step) via immunoprecipitation using the DNMT1-Trap.
说明

Bead size ~ 90 µm

实验时间 1.5 h
实验流程
  • Robust and versatile tool for biochemical analyses of human Dnmt1
  • Short incubation times (5 - 30 min)
  • Quantitative isolation of fusion proteins and transiently bound factors from cell extracts or organelles
  • Low unspecific binding
  • No contaminating heavy and light chains of conventional antibodies
试剂准备

Suggested buffer composition

  • Lysis buffer (CoIP): 10 mM Tris/Cl pH 7.5, 150 mM NaCl, 0.5 mM EDTA,0.5% NP-40
  • 10x RIPA buffer: 10 mM Tris/Cl pH 7.5, 150 mM NaCl, 5 mM EDTA, 0.1% SDS, 1% Triton X-100, 1% Deoxycholate
  • Dilution buffer: 10 mM Tris/Cl pH 7.5, 150 mM NaCl, 0.5 mM EDTA
  • Wash buffer: 10 mM Tris/Cl pH 7.5, 150 mM NaCl, 0.5 mM EDTA
  • Elution buffer: 200 mM glycine pH 2.5

实验流程
  • 1. For one immunoprecipitation reaction resuspend cell pellet (~10^7mammalian cells) in 200 µL lysis buffer by pipetting (or using a syringe).
    optional: add 1 mM PMSF and Protease inhibitor cocktail (not included) to lysis buffer
    optional for nuclear/chromatin proteins: add 1 mg/ml DNase and 2.5 mM MgCl2 (not included) to lysis buffer
  • 2. Place the tube on ice for 30 min with extensively pipetting every 10 min.
  • 3. Spin cell lysate at 20.000x g for 5 -10 minutes at 4°C.
  • 4. Transfer supernatant to a pre-cooled tube. Adjust volume with dilution buffer to 500 µL – 1000 µL. Discard pellet.
    optional: add 1 mM PMSF and Protease inhibitor cocktail (not included) to dilution buffer
    note: the cell lysate can be frozen at this point for long-term storage at -80°C For immunoblot analysis dilute 50 µL cell lysate with 50 µL 2x SDS-sample buffer(-> refer to as input).
  • 5. Equilibrate Dnmt1-Trap_A beads in dilution buffer. Resuspend 20 - 30 µL bead slurry in 500 µL ice cold dilution buffer and spin down at 2.500x g for 2 minutes at 4°C. Discard supernatant and wash beads 2 more times with 500 µL ice cold dilution buffer.
  • 6. Add cell lysate to equilibrated Dnmt1-Trap_A beads and incubate the Dnmt1-Trap_A beads with the cell lysate under constant mixing for 10 min – 2 h at room temperature or 4°C.
    note: during incubation of protein sample with the Dnmt1-Trap_A the final concentration of detergents should not exceed 0.2% to avoid unspecific binding to the matrix
  • 7. Spin tube at 2.500x g for 2 minutes at 4°C. For western blot analysis dilute 50 µL supernatant with 50 µL 2x SDS-sample buffer ( refer to as non-bound). Discard remaining supernatant.
  • 8. Wash beads two times with 500 µL ice cold wash buffer.
    optional: increase salt concentration in the second washing step up to 500 mM
  • 9. Dnmt1-Trap_A beads in 100 µL 2x SDS-Sample buffer or go to step 11.
  • 10. Boil resuspended beads for 10 minutes at 95°C to dissociate the immunocomplexes from the beads. The beads can be collected by centrifugation at 2.500x g for 2 minutes at 4°C and SDS-PAGE is performed with the supernatant (-> refer to as bound).
  • 11. optional: elute bound proteins by adding 50 µL 0.2 M glycine pH 2.5 (incubation time: 30 sec under constant mixing) followed by centrifugation. Transfer the supernatant to a fresh cup and add 5 µL 1M Tris base (pH 10.4) for neutralization. To increase elution efficiency this step can be repeated.
限制 仅限研究用
浓度 2.5 mL resin
缓冲液 20% EtOH
储存液 Sodium azide
注意事项 This product contains sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.
注意事项 Do not freeze.
储存条件 4 °C
有效期 12 months
厂商提供的图像
Immunoprecipitation (IP) image for Dnmt1-Trap A (ABIN1082205) Pulldown of Dnmt1 using the Dnmt1-Trap: Immunoprecipitations (IP) of Dnmt1 from a pr...
 image for Dnmt1-Trap A (ABIN1082205) On cell lysate: HEK 293T and HeLa lysate (size DNMT1: 183 kDa). Dilution 1:500. Secon...
Immunofluorescence (IF) image for Dnmt1-Trap A (ABIN1082205) Immunofluorescence: HeLa cells 3.7% PFA fixed. Primary antibody: 1/100 Dnmt1 antibody...
有引用在: Qin, Leonhardt, Pichler: "Regulation of DNA methyltransferase 1 by interactions and modifications." in: Nucleus (Austin, Tex.), Vol. 2, Issue 5, pp. 392-402, 2011 (PubMed).

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