Astana Biomed Group, an authorized Abcam distributor in Central Asia

Anti-ATF3 antibody [EPR22610-19] - BSA and Azide free (ab256534)

Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit monoclonal [EPR22610-19] to ATF3 - BSA and Azide free
  • Suitable for: IP, ChIP, WB, IHC-P, ICC/IF, Flow Cyt
  • Knockout validated
  • Reacts with: Mouse, Human

Overview

  • Product name

    Anti-ATF3 antibody [EPR22610-19] - BSA and Azide free
    See all ATF3 primary antibodies

  • Description

    Rabbit monoclonal [EPR22610-19] to ATF3 - BSA and Azide free

  • Host species

    Rabbit

  • Specificity

    IHC is not recommended for mouse.

  • Tested Applications & Species

    Application Species
    ChIP
    Human
    Flow Cyt
    Human
    ICC/IF
    Human
    IHC-P
    Human
    IP
    Mouse
    WB
    Human
    See all applications and species data

  • Immunogen

    Recombinant fragment. This information is proprietary to Abcam and/or its suppliers.

  • Positive control

    • WB: THP-1 treated with 80nM TPA overnight, then treated with 1µg/ml lipopolysaccharide (LPS) for 8 hours, whole cell lysate; RAW 264.7 (+/- treated with 1µg/ml lipopolysaccharide (LPS) for 2 hours) whole cell lysate; HCT116, A549, HepG2, HAP1 whole cell lysates. IHC-P: Human placenta and Hodgkin's lymphoma tissue. ICC/IF: THP-1 cells treated with PMA and LPS. Flow cyt: THP-1 cells treated with PMA and LPS. IP: RAW 264.7 (LPS -treated) whole cell lysate ChIP: Chromatin prepared from HeLa cells.

  • General notes

    Ab256534 is the carrier-free version of ab254268. This format is designed for use in antibody labeling, including fluorochromes, metal isotopes, oligonucleotides, enzymes.

     

    Our carrier-free formats are supplied in a buffer free of BSA, sodium azide and glycerol for higher conjugation efficiency.

    Use our conjugation kits  for antibody conjugates that are ready-to-use in as little as 20 minutes with

    ab256534 is compatible with the Maxpar® Antibody Labeling Kit from Fluidigm.

    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.

    The Life Science industry has been in the grips of a reproducibility crisis for a number of years. Abcam is leading the way in addressing the problem with our range of recombinant monoclonal antibodies and knockout edited cell lines for gold-standard validation.

    One factor contributing to the crisis is the use of antibodies that are not suitable. This can lead to misleading results and the use of incorrect data informing project assumptions and direction. To help address this challenge, we have introduced an application and species grid on our primary antibody datasheets to make it easy to simplify identification of the right antibody for your needs.

    Learn more here.

Properties

Images

  • Western blot - Anti-ATF3 antibody [EPR22610-19] - BSA and Azide free (ab256534)
    Western blot - Anti-ATF3 antibody [EPR22610-19] - BSA and Azide free (ab256534)
    All lanes : Anti-ATF3 antibody [EPR22610-19] - ChIP Grade (ab254268) at 1/1000 dilution

    Lane 1 : Wild-type A549 cell lysate
    Lane 2 : ATF3 knockout A549 cell lysate

    Lysates/proteins at 20 µg per lane.

    Performed under reducing conditions.

    Predicted band size: 21 kDa
    Observed band size: 21 kDa



    This data was developed using the same antibody clone in a different buffer formulation (ab254268).

    Lanes 1-2: Merged signal (red and green). Green - ab254268 observed at 21 kDa. Red - loading control ab8245 observed at 37 kDa.

     ab254268 Recombinant Anti-ATF3 antibody [EPR22610-19] was shown to specifically react with ATF3 in wild-type A549 cells. Loss of signal was observed when knockout cell line ab266955 (knockout cell lysate ab257075) was used. Wild-type and ATF3 knockout samples were subjected to SDS-PAGE. ab254268 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

     

     

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ATF3 antibody [EPR22610-19] - BSA and Azide free (ab256534)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ATF3 antibody [EPR22610-19] - BSA and Azide free (ab256534)

    Immunohistochemical analysis of paraffin-embedded Human placenta tissue labeling ATF3 with ab254268 at 1/500 dilution (1.14 ug/ml) followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Nuclear staining in human placenta (PMID/ 28947613). Counterstained with Hematoxylin. Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab254268).

  • Immunocytochemistry/ Immunofluorescence - Anti-ATF3 antibody [EPR22610-19] - BSA and Azide free (ab256534)
    Immunocytochemistry/ Immunofluorescence - Anti-ATF3 antibody [EPR22610-19] - BSA and Azide free (ab256534)

    Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized THP-1 (human monocytic leukemia monocyte) cells labelling ATF3 with ab254268 at 1/100 (5.7 ug/ml) dilution, followed by ab150077 AlexaFluor® 488 Goat anti-Rabbit secondary antibody at 1/1000 (5.7 ug/ml) dilution (Green). Confocal image showing nuclear staining in THP-1 cells treated with Phorbol 12-myristate 13-acetate (80 nM) for 16 h, then along with lipopolysaccharides (1ug/ml) for 8 h. ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is ab150077 AlexaFluor® 488 Goat anti-Rabbit secondary at 1/1000 (2 ug/ml) dilution.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab254268).

  • Flow Cytometry - Anti-ATF3 antibody [EPR22610-19] - BSA and Azide free (ab256534)
    Flow Cytometry - Anti-ATF3 antibody [EPR22610-19] - BSA and Azide free (ab256534)

    Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized THP-1 (human monocytic leukemia monocyte) treated with 80nM Phorbol 12-myristate 13-acetate (PMA) for 16h, then together with 1μg/ml lipopolysaccharides (LPS) for 8h (Red) / Untreated control (Green) cells labelling ATF3 with ab254268 at 1/600 compared with a Rabbit monoclonal IgG (ab172730) isotype control (Black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab254268).

  • Western blot - Anti-ATF3 antibody [EPR22610-19] - BSA and Azide free (ab256534)
    Western blot - Anti-ATF3 antibody [EPR22610-19] - BSA and Azide free (ab256534)
    All lanes : Anti-ATF3 antibody [EPR22610-19] - ChIP Grade (ab254268) at 1/1000 dilution

    Lane 1 : Wild-type HCT116 cell lysate
    Lane 2 : ATF3 knockout HCT116 cell lysate

    Lysates/proteins at 20 µg per lane.

    Performed under reducing conditions.

    Predicted band size: 21 kDa
    Observed band size: 21 kDa



    This data was developed using the same antibody clone in a different buffer formulation (ab254268).

    Lanes 1-2: Merged signal (red and green). Green - ab254268 observed at 21 kDa. Red - loading control ab8245 observed at 37 kDa.

     ab254268 Recombinant Anti-ATF3 antibody [EPR22610-19] was shown to specifically react with ATF3 in wild-type HCT116 cells. Loss of signal was observed when knockout cell line ab266872 (knockout cell lysate ab257074) was used. Wild-type and ATF3 knockout samples were subjected to SDS-PAGE. ab254268 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

     

     

  • Western blot - Anti-ATF3 antibody [EPR22610-19] - BSA and Azide free (ab256534)
    Western blot - Anti-ATF3 antibody [EPR22610-19] - BSA and Azide free (ab256534)
    All lanes : Anti-ATF3 antibody [EPR22610-19] - ChIP Grade (ab254268) at 1/1000 dilution

    Lane 1 : 293T (Human embryonic kidney epithelial cell) whole cell lysate
    Lane 2 : Human liver tissue lysate
    Lane 3 : Raw264.7 (Mouse abelson murine leukemia virus-induced tumor macrophage) whole cell lysate
    Lane 4 : Mouse liver tissue lysate
    Lane 5 : MEF (Mouse embryonic fibroblast (immortalized)) whole cell lysate
    Lane 6 : Mouse heart tissue 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: 21 kDa
    Observed band size: 21 kDa


    Exposure time: 180 seconds


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

    Rabbit monoclonal [EPR16891] to GAPDH (ab181602) used as loading control.

    ATF3 has a low expression level in some cell lines and tissues, but is increased under treatment (PMID: 8622660, PMID: 22053207, PMID: 20018623, PMID: 29940414).

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab254268).

  • Western blot - Anti-ATF3 antibody [EPR22610-19] - BSA and Azide free (ab256534)
    Western blot - Anti-ATF3 antibody [EPR22610-19] - BSA and Azide free (ab256534)
    All lanes : Anti-ATF3 antibody [EPR22610-19] - ChIP Grade (ab254268) at 1/1000 dilution

    Lane 1 : HepG2 (human hepatocellular carcinoma epithelial cell), whole cell lysate
    Lane 2 : HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate
    Lane 3 : HEK-293T (human embryonic kidney epithelial cell), whole cell lysate
    Lane 4 : Daudi (human Burkitts lymphoma lymphoblast), whole cell lysate
    Lane 5 : Wild-type HAP1 whole cell lysate
    Lane 6 : ATF3 knockout HAP1 whole cell lysate

    Lysates/proteins at 20 µg per lane.

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

    Predicted band size: 21 kDa
    Observed band size: 21 kDa


    Exposure time: 26 seconds


    Blocking and dilution buffer: 5% NFDM/TBST.

    Negative control: Daudi (PMID:19136462). 

    The molecular weight observed is consistent with what has been described in the literature (PMID:18692824).

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab254268).

     

  • ChIP - Anti-ATF3 antibody [EPR22610-19] - BSA and Azide free (ab256534)
    ChIP - Anti-ATF3 antibody [EPR22610-19] - BSA and Azide free (ab256534)

    Chromatin was prepared from HeLa cells according to the Abcam Dual X-ChIP protocol. Cells were fixed with EGS for 30min, then formaldehyde for 10min. The ChIP was performed with 25 μg of chromatin, 5 μg of ab254268 (red), and 20 μl of Protein A/G sepharose beads. 5 μg of rabbit normal IgG was added to the beads control (gray). The immunoprecipitated DNA was quantified by real time PCR (sybr green approach). Primers and probes are located in the first kb of the transcribed region.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab254268).

  • Immunoprecipitation - Anti-ATF3 antibody [EPR22610-19] - BSA and Azide free (ab256534)
    Immunoprecipitation - Anti-ATF3 antibody [EPR22610-19] - BSA and Azide free (ab256534)

    ATF3 was immunoprecipitated from 0.35 mg RAW264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) treated with 1ug/ml lipopolysaccharide (LPS) for 2h whole cell lysate 10ug with ab254268 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab254268 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/1000 dilution.

    Lane 1: RAW264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) treated with 1ug/ml lipopolysaccharide (LPS) for 2h whole cell lysate  10ug.

    Lane 2: ab254268 IP in RAW264.7 treated with 1ug/ml lipopolysaccharide (LPS) for 2h whole cell lysate.

    Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab254268 in RAW264.7 treated with 1ug/ml lipopolysaccharide (LPS) for 2h whole cell lysate.

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

    Exposure time: 6 seconds.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab254268).

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ATF3 antibody [EPR22610-19] - BSA and Azide free (ab256534)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ATF3 antibody [EPR22610-19] - BSA and Azide free (ab256534)

    Immunohistochemical analysis of paraffin-embedded Human Hodgkin's lymphoma tissue labeling ATF3 with ab254268 at 1/500 dilution (1.14 ug/ml) followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Nuclear staining in Reed-Sternberg (HRS) cells of human (PMID: 16263788) is observed. Counterstained with Hematoxylin. Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab254268).

  • Anti-ATF3 antibody [EPR22610-19] - BSA and Azide free (ab256534)
    Anti-ATF3 antibody [EPR22610-19] - BSA and Azide free (ab256534)

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