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Validated using a knockout cell lineRecombinantRabMAb

Recombinant Anti-EED antibody [EPR23043-5] - BSA and Azide free (ab264566)

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Western blot - Anti-EED antibody [EPR23043-5] - BSA and Azide free (ab264566)
  • Western blot - Anti-EED antibody [EPR23043-5] - ChIP Grade – BSA and Azide free (ab264566)
  • Immunoprecipitation - Anti-EED antibody [EPR23043-5] - ChIP Grade – BSA and Azide free (ab264566)
  • Western blot - Anti-EED antibody [EPR23043-5] - ChIP Grade – BSA and Azide free (ab264566)
  • Immunoprecipitation - Anti-EED antibody [EPR23043-5] - BSA and Azide free (ab264566)
  • ChIP - Anti-EED antibody [EPR23043-5] - BSA and Azide free (ab264566)

Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit monoclonal [EPR23043-5] to EED - BSA and Azide free
  • Suitable for: IP, WB, ChIP
  • Knockout validated
  • Reacts with: Mouse, Human

Conjugates logo Related conjugates and formulations

Unconjugated

Overview

  • Product name

    Anti-EED antibody [EPR23043-5] - BSA and Azide free
    See all EED primary antibodies

  • Description

    Rabbit monoclonal [EPR23043-5] to EED - BSA and Azide free

  • Host species

    Rabbit

  • Specificity

    ab240650 detects an unknown band close to the target bands in cytoplasm.

  • Tested applications

    Suitable for: IP, WB, ChIPmore details
    Unsuitable for: ICC/IF or IHC-P

  • Species reactivity

    Reacts with: Mouse, Human

  • Immunogen

    This product was produced with the following immunogens:
    Synthetic peptide. This information is proprietary to Abcam and/or its suppliers.

    Synthetic peptide. This information is proprietary to Abcam and/or its suppliers.

  • Positive control

    • WB: Wild type HAP1, K562, 293T, NIH/3T3 and C2C12 lysates. IP: K562 cells.

  • General notes

    ab264566 is the carrier-free version of ab240650.

    Our carrier-free antibodies are typically supplied in a PBS-only formulation, purified and free of BSA, sodium azide and glycerol. The carrier-free buffer and high concentration allow for increased conjugation efficiency.

    This conjugation-ready format is designed for use with fluorochromes, metal isotopes, oligonucleotides, and enzymes, which makes them ideal for antibody labelling, functional and cell-based assays, flow-based assays (e.g. mass cytometry) and Multiplex Imaging applications.

    Use our conjugation kits for antibody conjugates that are ready-to-use in as little as 20 minutes with <1 minute hands-on-time and 100% antibody recovery: available for fluorescent dyes, HRP, biotin and gold.

    This product is compatible with the Maxpar® Antibody Labeling Kit from Fluidigm, without the need for antibody preparation. Maxpar® is a trademark of Fluidigm Canada Inc.

    This product is a recombinant monoclonal antibody, which offers several advantages including:

    • - High batch-to-batch consistency and reproducibility
    • - Improved sensitivity and specificity
    • - Long-term security of supply
    • - Animal-free production
    For more information see here.

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.

Properties

Applications

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab264566 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
IP
Use at an assay dependent concentration.

 

 
WB
Use at an assay dependent concentration. Predicted molecular weight: 50 kDa.

 

 
ChIP
Use at an assay dependent concentration.

 

 
Notes
IP
Use at an assay dependent concentration.

 

 
WB
Use at an assay dependent concentration. Predicted molecular weight: 50 kDa.

 

 
ChIP
Use at an assay dependent concentration.

 

 
Application notes
Is unsuitable for ICC/IF or IHC-P.

Target

  • Function

    Polycomb group (PcG) protein. Component of the PRC2/EED-EZH2 complex, which methylates 'Lys-9' and 'Lys-27' of histone H3, leading to transcriptional repression of the affected target gene. The PRC2/EED-EZH2 complex may also serve as a recruiting platform for DNA methyltransferases, thereby linking two epigenetic repression systems. Genes repressed by the PRC2/EED-EZH2 complex include HOXC8, HOXA9, MYT1 and CDKN2A.

  • Tissue specificity

    Expressed in brain, colon, heart, kidney, liver, lung, muscle, ovary, peripheral blood leukocytes, pancreas, placenta, prostate, spleen, small intestine, testis, thymus and uterus. Appears to be overexpressed in breast and colon cancer.

  • Sequence similarities

    Belongs to the WD repeat ESC family.
    Contains 7 WD repeats.

  • Developmental stage

    Expression peaks at the G1/S phase boundary.

  • Cellular localization

    Nucleus. Chromosome. Transiently colocalizes with XIST at inactive X chromosomes.
  • Information by UniProt

  • Database links

    • Alternative names

      • eed antibody
      • EED protein antibody
      • EED, mouse, homolog of antibody
      • EED_HUMAN antibody
      • Embryonic ectoderm development antibody
      • Embryonic ectoderm development isoform a antibody
      • Embryonic ECTODERM development protein homolog antibody
      • Embryonic ectoderm development protein, mouse, homolog of antibody
      • hEED antibody
      • OTTHUMP00000235483 antibody
      • OTTHUMP00000235484 antibody
      • OTTHUMP00000235485 antibody
      • Polycomb protein eed antibody
      • WAIT 1 antibody
      • WAIT-1 antibody
      • WAIT1 antibody
      • WD protein associating with integrin cytoplasmic tails 1 antibody
      see all

    Images

    • Western blot - Anti-EED antibody [EPR23043-5] - BSA and Azide free (ab264566)
      Western blot - Anti-EED antibody [EPR23043-5] - BSA and Azide free (ab264566)
      All lanes : Anti-EED antibody [EPR23043-5] - ChIP Grade (ab240650) at 1/1000 dilution

      Lane 1 : Wild type HAP1 whole cell lysate
      Lane 2 : EED knockout HAP1 whole cell lysate
      Lane 3 : Wild type HAP1 nuclear fraction lysate
      Lane 4 : EED knockout HAP1 nuclear fraction lysate
      Lane 5 : Wild type HAP1 cytoplasmic lysate
      Lane 6 : EED knockout HAP1 cytoplasmic lysate

      Lysates/proteins at 20 µg per lane.

      Secondary
      All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

      Predicted band size: 50 kDa
      Observed band size: 50-70 kDa why is the actual band size different from the predicted?


      Exposure time: 3 seconds


      This data was developed using ab240650, the same antibody clone in a different buffer formulation. 

      Blocking and dilution buffer and concentration: 5% NFDM/TBST

      ab240650 detects an unknown band close to the target bands in cytoplasm.

    • Western blot - Anti-EED antibody [EPR23043-5] - ChIP Grade – BSA and Azide free (ab264566)
      Western blot - Anti-EED antibody [EPR23043-5] - ChIP Grade – BSA and Azide free (ab264566)
      All lanes : Anti-EED antibody [EPR23043-5] - ChIP Grade (ab240650) at 1/1000 dilution

      Lanes 1-2 : 293T (human embryonic kidney epithelial cell) whole cell lysate
      Lane 3 : K562 (human chronic myelogenous leukemia lymphoblast), whole cell lysate
      Lane 4 : 293T (human embryonic kidney epithelial cell), whole cell lysate
      Lane 5 : NIH/3T3 (mouse embryonic fibroblast), whole cell lysate
      Lane 6 : C2C12 (mouse myoblasts myoblast), whole cell lysate

      Secondary
      All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/20000 dilution

      Predicted band size: 50 kDa



      This data was developed using ab240650, the same antibody clone in a different buffer formulation. 

      Blocking and diluting buffer and concentration: ab240650 was shown to specifically react with EED in wild-type HAP1 cells as signal was lost in EED knockout cells. Wild-type and EED knockout samples were subjected to SDS-PAGE. ab240650 and ab181602 (Rabbit anti-GAPDH loading control) were incubated 1 hour at room temperature at 1/1000 dilution and 1/200, 000 dilution respectively. Blots were developed with Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) secondary antibody at 1/100, 000 dilution for 1 hour at room temperature before imaging.

       

      The molecular weight observed is consistent with what has been described in the literature (PMID:27578866, 9584199). EED cDNA encodes 441-aa-long protein and 535-aa-long protein.

       

      Lanes 1-3: 15 seconds; Lanes 4-6: 37 seconds.

      Exposure time: 

    • Immunoprecipitation - Anti-EED antibody [EPR23043-5] - ChIP Grade – BSA and Azide free (ab264566)
      Immunoprecipitation - Anti-EED antibody [EPR23043-5] - ChIP Grade – BSA and Azide free (ab264566)

      This data was developed using ab240650, the same antibody clone in a different buffer formulation.

      EED was immunoprecipitated from 0.35 mg K562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate 10ug with ab240650 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab240650 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

      Lane 1: K562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate 10ug

      Lane 2: ab240650 IP in K562 whole cell lysate

      Lane 3:Rabbit monoclonal IgG (ab172730) instead of ab240650 in K562 whole cell lysate

      Blocking and dilution buffer and concentration: 5% NFDM/TBST.

      Exposure time: 1 minutes

    • Western blot - Anti-EED antibody [EPR23043-5] - ChIP Grade – BSA and Azide free (ab264566)
      Western blot - Anti-EED antibody [EPR23043-5] - ChIP Grade – BSA and Azide free (ab264566)
      All lanes : Anti-EED antibody [EPR23043-5] - ChIP Grade (ab240650) at 1/1000 dilution

      Lane 1 : Wild type HAP1 whole cell lysate
      Lane 2 : EED knockout HAP1 whole cell lysate
      Lane 3 : K562 (human chronic myelogenous leukemia lymphoblast), whole cell lysate
      Lane 4 : 293T (human embryonic kidney epithelial cell), whole cell lysate
      Lane 5 : NIH/3T3 (mouse embryonic fibroblast), whole cell lysate
      Lane 6 : C2C12 (mouse myoblasts myoblast), whole cell lysate

      Lysates/proteins at 20 µg per lane.

      Secondary
      All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

      Predicted band size: 50 kDa
      Observed band size: 50-70 kDa why is the actual band size different from the predicted?



      Blocking and diluting buffer and concentration: 5% NFDM/TBST ab240650 was shown to specifically react with EED in wild-type HAP1 cells as signal was lost in EED knockout cells. Wild-type and EED knockout samples were subjected to SDS-PAGE. ab240650 and ab181602 (Rabbit anti-GAPDH loading control) were incubated 1 hour at room temperature at 1/1000 dilution and 1/200,000 dilution respectively. Blots were developed with Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) secondary antibody at 1/100,000 dilution for 1 hour at room temperature before imaging.  The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID:27578866, 9584199). EED cDNA encodes 441-aa-long protein and 535-aa-long protein. 

      Exposure time: Lanes 1-3: 15 seconds Lanes 4-6: 37 seconds

      This data was developed using the same antibody clone in a different buffer formulation containing PBS, glycerol, azide and BSA (ab240650).

    • Immunoprecipitation - Anti-EED antibody [EPR23043-5] - BSA and Azide free (ab264566)
      Immunoprecipitation - Anti-EED antibody [EPR23043-5] - BSA and Azide free (ab264566)

      EED was immunoprecipitated from 0.35 mg K562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate with ab240650 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab240650 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/5000 dilution.

      Lane 1: K562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate 10 µg

      Lane 2: ab240650 IP in K562 whole cell lysate

      Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab240650 in K562 whole cell lysate

      Blocking and dilution buffer and concentration: 5% NFDM/TBST.

      Exposure time: 1 min

      This data was developed using the same antibody clone in a different buffer formulation containing PBS, glycerol, azide and BSA (ab240650).

    • ChIP - Anti-EED antibody [EPR23043-5] - BSA and Azide free (ab264566)
      ChIP - Anti-EED antibody [EPR23043-5] - BSA and Azide free (ab264566)

      Chromatin was prepared from NT2/D1 cells according to the Abcam Dual-X-ChIP protocol*. Cells were fixed with 1.5 mM EGS for 30mins and then formaldehyde for 10min.

      The ChIP was performed with 25 µg of chromatin, 5 µg of ab240650 (red), or 5 µg of rabbit normal IgG ab172730 (gray) and 20 µl of Protein A/G sepharose beads.  The immunoprecipitated DNA was quantified by real time PCR (Taqman approach for active and inactive loci, Sybr green approach for heterochromatic loci).

      Primers and probes are commercial primers from Millipore (Cat. No.: 17-10034) and CST (85322S)

      *https://www.abcam.com/resources?keywords=X%20ChIP%20protocol

      This data was developed using the same antibody clone in a different buffer formulation containing PBS, glycerol, azide and BSA (ab240650).

    Protocols

    To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.

    Click here to view the general protocols

    References (0)

    Publishing research using ab264566? Please let us know so that we can cite the reference in this datasheet.

    ab264566 has not yet been referenced specifically in any publications.

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