Overview

  • Product name

    Anti-ATF6 antibody [EPR22690-84] - ChIP Grade
    See all ATF6 primary antibodies
  • Description

    Rabbit monoclonal [EPR22690-84] to ATF6 - ChIP Grade
  • Host species

    Rabbit
  • Tested applications

    Suitable for: WB, IHC-P, ChIP, IPmore details
    Unsuitable for: Flow Cyt or ICC/IF
  • Species reactivity

    Reacts with: Human
  • Immunogen

    Recombinant fragment within Human ATF6 aa 1-300. The exact sequence is proprietary.
    Database link: P18850

  • Positive control

    • WB: HAP1 and HeLa whole cell lysates. IHC-P: Human kidney tissue. IP: HeLa whole cell lysate. ChIP: Chromatin prepared from RAW 264.7 (treated with tunicamycin) and HeLa (treated with thapsigargin) cells.
  • General notes

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

    This product is a recombinant rabbit monoclonal antibody.

Properties

  • Form

    Liquid
  • Storage instructions

    Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C long term. Avoid freeze / thaw cycle.
  • Storage buffer

    Preservative: 0.01% Sodium azide
    Constituents: PBS, 40% Glycerol, 0.05% BSA
  • Concentration information loading...
  • Purity

    Protein A purified
  • Clonality

    Monoclonal
  • Clone number

    EPR22690-84
  • Isotype

    IgG
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab227830 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
WB 1/1000. Predicted molecular weight: 74 kDa.
IHC-P 1/1000. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
ChIP Use 5 µg for 25 µg of chromatin.
IP 1/30.
  • Application notes
    Is unsuitable for Flow Cyt or ICC/IF.
  • Target

    • Function

      Transcription factor that acts during endoplasmic reticulum stress by activating unfolded protein response target genes. Binds DNA on the 5'-CCAC[GA]-3'half of the ER stress response element (ERSE) (5'-CCAAT-N(9)-CCAC[GA]-3') and of ERSE II (5'-ATTGG-N-CCACG-3'). Binding to ERSE requires binding of NF-Y to ERSE. Could also be involved in activation of transcription by the serum response factor.
    • Tissue specificity

      Ubiquitous.
    • Sequence similarities

      Belongs to the bZIP family. ATF subfamily.
      Contains 1 bZIP domain.
    • Domain

      The basic domain functions as a nuclear localization signal.
      The basic leucine-zipper domain is sufficient for association with the NF-Y trimer and binding to ERSE.
    • Post-translational
      modifications

      During unfolded protein response an approximative 50 kDa fragment containing the cytoplasmic transcription factor domain is released by proteolysis. The cleavage seems to be performed sequentially by site-1 and site-2 proteases.
      N-glycosylated. The glycosylation status may serve as a sensor for ER homeostasis, resulting in ATF6 activation to trigger the unfolded protein response (UPR).
      Phosphorylated in vitro by MAPK14/P38MAPK.
    • Cellular localization

      Endoplasmic reticulum membrane and Nucleus. Under ER stress the cleaved N-terminal cytoplasmic domain translocates into the nucleus.
    • Information by UniProt
    • Database links

    • Alternative names

      • Activating transcription factor 6 alpha antibody
      • Activating transcription factor 6 antibody
      • ATF 6 antibody
      • ATF6 alpha antibody
      • ATF6 antibody
      • ATF6-alpha antibody
      • ATF6A antibody
      • ATF6A_HUMAN antibody
      • cAMP dependent transcription factor ATF 6 alpha antibody
      • cAMP-dependent transcription factor ATF-6 alpha antibody
      • Cyclic AMP dependent transcription factor ATF 6 alpha antibody
      • DKFZp686P2194 antibody
      • ESTM49 antibody
      • FLJ21663 antibody
      • Processed cyclic AMP dependent transcription factor ATF 6 alpha antibody
      • Processed cyclic AMP-dependent transcription factor ATF-6 alpha antibody
      see all

    Images

    • All lanes : Anti-ATF6 antibody [EPR22690-84] - ChIP Grade (ab227830) at 1/1000 dilution

      Lane 1 : Wild type HAP1 whole cell lysate 20 µg
      Lane 2 : ATF6 knockout HAP1 whole cell lysate 20 µg

      Secondary
      All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

      Predicted band size: 74 kDa
      Observed band size: 90 kDa
      why is the actual band size different from the predicted?



      ab227830 was shown to specifically react with ATF6 in wild-type HAP1 cells as signal was lost in ATF6 knockout cells. Wild-type and ATF6 knockout samples were subjected to SDS-PAGE. ab227830 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 1/20,000 dilution for 1 hour at room temperature before imaging.
       
      Exposure time 62 seconds.
       
      Blocking/Diluting buffer and concentration: 5% NFDM/TBST.

       

    • All lanes : Anti-ATF6 antibody [EPR22690-84] - ChIP Grade (ab227830) at 1/1000 dilution

      Lane 1 : Untreated HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate
      Lane 2 : HeLa treated with 1 µ? thapsigargin for 1 hour, whole cell lysate

      Lysates/proteins at 10 µg per lane.

      Secondary
      All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

      Predicted band size: 74 kDa
      Observed band size: 50,90 kDa why is the actual band size different from the predicted?



      ATF6 is cleaved upon ER stress and the molecular weight observed is consistent with what has been described in the literature (PMID: 25149687; 11163209).
       
      Exposure time 3 minutes.
       
      Blocking and diluting buffer and concentration: 5% NFDM/TBST.
    • ATF6 was immunoprecipitated from 0.35 mg HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate with ab227830 at 1/30 dilution (2μg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab227830 1/1000 dilution (0.5 μg/ml). VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/5000 dilution.

      Lane 1: HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate 10μg

      Lane 2: ab254324 IP in HeLa whole cell lysate

      Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab227830 in HeLa whole cell lysate.

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

      Exposure time: 3 min.

      Lysate were made freshly and used in IP test immediately to minimize protein degradation. Incubation time was 2h.

    • Immunohistochemical analysis of paraffin-embedded Human kidney tissue labeling ATF6 with ab227830 at 1/1000 dilution (0.566 μg/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on human kidney (PMID: 25725420) is observed. The section was incubated with ab227830 for 30 mins at room temperature.

      The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20mins.

      Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

    • Chromatin was prepared from HeLa cells treated with thapsigargin (1 uM, 1 h) according to the Abcam Dual-X-ChIP protocol*. Cells were fixed with 1.5 mM EGS for 30 mins and then formaldehyde for 10 min.
      The ChIP was performed with 25 µg of chromatin, 5 µg of ab227830 (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 from paper PMID: 17535801.


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

       

    • Chromatin was prepared from RAW 264.7 cells treated with tunicamycin (5 ug/ml, 4 h) according to the Abcam Dual-X-ChIP protocol*. Cells were fixed with 1.5 mM EGS for 30 mins and then formaldehyde for 10 min.
      The ChIP was performed with 25 µg of chromatin, 5 µg of ab227830 (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 from paper PMCID: PMC5179193.


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

    References

    ab227830 has not yet been referenced specifically in any publications.

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