Name: Anti-ATF3 antibody [EPR22610-19] - ChIP Grade
SKU: ab254268
Rating: 3 (2 reviews)

Rabbit Recombinant Monoclonal ATF3 antibody. Suitable for ChIC/CUT&RUN-seq, ChIP, Flow Cyt (Intra), ICC/IF, WB, IP, IHC-P and reacts with Human, Mouse samples. Cited in 19 publications.

Images

Western blot - Anti-ATF3 antibody [EPR22610-19] - ChIP Grade (AB254268), expandable thumbnail

Publications

Key facts

Isotype: IgG
Host species: Rabbit
Storage buffer: pH: 7.2 - 7.4
Preservative: 0.01% Sodium azide
Constituents: PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA
Form: Liquid
Clonality: Monoclonal

Immunogen

The exact immunogen used to generate this antibody is proprietary information.

Reactivity data

Select an application

Product promiseTestedExpectedPredictedNot recommended

	ChIC/CUT&RUN-seq	ChIP	Flow Cyt (Intra)	ICC/IF	WB	IP	IHC-P
Human	Tested	Tested	Tested	Tested	Tested	Expected	Tested
Mouse	Expected	Expected	Expected	Expected	Tested	Tested	Expected

Tested
Tested

Species	Dilution info	Notes
Species Human	Dilution info -	Notes -

Expected
Expected

Species	Dilution info	Notes
Species Mouse	Dilution info Use at an assay dependent concentration.	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Human	Dilution info 5 µg for 25 µg chromatin	Notes -

Expected
Expected

Species	Dilution info	Notes
Species Mouse	Dilution info Use at an assay dependent concentration.	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Human	Dilution info 1/600	Notes -

Expected
Expected

Species	Dilution info	Notes
Species Mouse	Dilution info Use at an assay dependent concentration.	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Human	Dilution info 1/100	Notes -

Expected
Expected

Species	Dilution info	Notes
Species Mouse	Dilution info Use at an assay dependent concentration.	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Mouse	Dilution info 1/1000	Notes -
Species Human	Dilution info 1/1000	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Mouse	Dilution info 1/30	Notes -

Expected
Expected

Species	Dilution info	Notes
Species Human	Dilution info Use at an assay dependent concentration.	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Human	Dilution info 1/500	Notes IHC is not recommended for mouse. Perform heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Expected
Expected

Species	Dilution info	Notes
Species Mouse	Dilution info Use at an assay dependent concentration.	Notes -

Associated Products

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Target data

See full target information ATF3

Function

This protein binds the cAMP response element (CRE) (consensus: 5'-GTGACGT[AC][AG]-3'), a sequence present in many viral and cellular promoters. Represses transcription from promoters with ATF sites. It may repress transcription by stabilizing the binding of inhibitory cofactors at the promoter. Isoform 2. Activates transcription presumably by sequestering inhibitory cofactors away from the promoters. Isoform 3. Stress-induced isoform, counteracts the transcriptional repression of isoform 1.

Alternative names

Cyclic AMP-dependent transcription factor ATF-3, cAMP-dependent transcription factor ATF-3, Activating transcription factor 3, ATF3

Recommended products

Rabbit Recombinant Monoclonal ATF3 antibody. Suitable for ChIC/CUT&RUN-seq, ChIP, Flow Cyt (Intra), ICC/IF, WB, IP, IHC-P and reacts with Human, Mouse samples. Cited in 19 publications.

Key facts

Isotype

IgG

Host species

Rabbit

Storage buffer

pH: 7.2 - 7.4
Preservative: 0.01% Sodium azide
Constituents: PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA

Form

Liquid

Clonality

Monoclonal

Immunogen

The exact immunogen used to generate this antibody is proprietary information.

Clone number

EPR22610-19

Purification technique

Affinity purification Protein A

Specificity

IHC is not recommended for mouse.

Stimulation may be required to allow detection of the target protein due to low levels of endogenous expression in some samples. Please see images below for recommended treatment conditions and positive controls.

ATF3 has a low expression level in some cell lines and tissues, but is increased under treatment (PMID: 8622660, 22053207, 20018623, 29940414, and 27912076).

Concentration

Storage

Shipped at conditions: Blue Ice
Appropriate short-term storage duration: 1-2 weeks
Appropriate short-term storage conditions: +4°C
Appropriate long-term storage conditions: -20°C
Aliquoting information: Upon delivery aliquot
Storage information: Avoid freeze / thaw cycle

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 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 batch production

For more information, read more on recombinant antibodies.

Supplementary info

This supplementary information is collated from multiple sources and compiled automatically.

Activity summary

ATF3 also known as Activating Transcription Factor 3 functions primarily as a stress-responsive transcription factor. This protein has a molecular weight of about 21 kDa and is part of the ATF/CREB family of transcription factors. ATF3 is expressed in various tissues and is known for its role in stress response and regulation of gene expression. The expression levels change in response to cellular stress and it may interact with several other transcription factors to modulate gene expression profiles.

Biological function summary

ATF3 acts as a regulator of cellular stress responses and can influence cell cycle arrest and apoptosis. It does not form a large complex but interacts with diverse partners to execute its functions. Studies often use HCT116 cells to investigate ATF3's role where it can serve as a marker of cellular stress and inflammation. The protein regulates genes involved in maintaining homeostasis in stressed cells.

Pathways

ATF3 is involved in stress response and apoptotic pathways. In the context of apoptosis ATF3 interacts with proteins like p53 and Jun which are important for inducing cell cycle arrest and programmed cell death. In stress response pathways ATF3 modifies transcriptional activity in reaction to various stress signals providing a checkpoint for damaged cells before repairing or entering programmed death.

Associated diseases and disorders

ATF3 relates to cancer and cardiovascular diseases. In cancer abnormal expression of ATF3 can influence tumor growth and metastasis often acting through its interactions with proteins like NF-kB. Additionally in cardiovascular disorders ATF3's role in regulating stress responses underpins its involvement in conditions linked to inflammation and atherosclerosis again with NF-kB signaling being a relevant pathway of interaction. Researchers continue to study ATF3 to uncover therapeutic targets for these and other diseases.

Product promise

We are dedicated to supporting your work with high quality reagents and we are here for you every step of the way should you need us.

In the unlikely event of one of our products not working as expected, you are covered by our product promise.

Full details and terms and conditions can be found here:
Terms & Conditions.

13 product images

Western blot - Anti-ATF3 antibody [EPR22610-19] - ChIP Grade (ab254268)
Lanes 1-2: Merged signal (red and green). Green - ab254268 observed at 21 kDa. Red - loading control Anti-GAPDH antibody [6C5] - 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 Human ATF3 knockout HCT116 cell line ab266872 (knockout cell lysate Human ATF3 knockout HCT116 cell lysate ab257074) was used. Wild-type and ATF3 knockout samples were subjected to SDS-PAGE. ab254268 and Anti-GAPDH antibody [6C5] - Loading Control (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 (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (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.
All lanes: Western blot - Anti-ATF3 antibody [EPR22610-19] - ChIP Grade (ab254268) at 1/1000 dilution
Lane 1: Wild-type HCT116 cell lysate at 20 µg
Lane 2: ATF3 knockout HCT116 cell lysate at 20 µg
Lane 2: Western blot - Human ATF3 knockout HCT116 cell line (Human ATF3 knockout HCT116 cell line ab266872)
Performed under reducing conditions.
Predicted band size: 21 kDa
Observed band size: 21 kDa
ChIP - Anti-ATF3 antibody [EPR22610-19] - ChIP Grade (ab254268)
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.
Immunocytochemistry/ Immunofluorescence - Anti-ATF3 antibody [EPR22610-19] - ChIP Grade (ab254268)
ab254268 staining ATF3 in wild-type Hap1 cells (top panel) and ATF3 knockout Hap1 cells (bottom panel). The cells were fixed with 4% paraformaldehyde (10 min) permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab254268 at 1/100 dilution and Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291 (Tubulin) at 1/1000 dilution overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to rabbit IgG (Alexa Fluor® 488) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to mouse IgG (Alexa Fluor® 594) (Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120) at 2 μg/ml (shown in pseudo color red). Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).
Western blot - Anti-ATF3 antibody [EPR22610-19] - ChIP Grade (ab254268)
Lanes 1-2: Merged signal (red and green). Green - ab254268 observed at 21 kDa. Red - loading control Anti-GAPDH antibody [6C5] - 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 Human ATF3 knockout A549 cell line ab266955 (knockout cell lysate Human ATF3 knockout A549 cell lysate ab257075) was used. Wild-type and ATF3 knockout samples were subjected to SDS-PAGE. ab254268 and Anti-GAPDH antibody [6C5] - Loading Control (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 (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (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.
All lanes: Western blot - Anti-ATF3 antibody [EPR22610-19] - ChIP Grade (ab254268) at 1/1000 dilution
Lane 1: Wild-type A549 cell lysate at 20 µg
Lane 2: ATF3 knockout A549 cell lysate at 20 µg
Lane 2: Western blot - Human ATF3 knockout A549 cell line (Human ATF3 knockout A549 cell line ab266955)
Performed under reducing conditions.
Predicted band size: 21 kDa
Observed band size: 21 kDa
Western blot - Anti-ATF3 antibody [EPR22610-19] - ChIP Grade (ab254268)
Blocking/Diluting buffer and concentration: 5% NFDM/TBST.
Rabbit monoclonal [EPR16891] to GAPDH (Anti-GAPDH antibody [EPR16891] - Loading Control 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).
All lanes: Western blot - Anti-ATF3 antibody [EPR22610-19] - ChIP Grade (ab254268) at 1/1000 dilution
Lane 1: 293T (Human embryonic kidney epithelial cell) whole cell lysate at 20 µg
Lane 2: Human liver tissue lysate at 20 µg
Lane 3: Raw264.7 (Mouse abelson murine leukemia virus-induced tumor macrophage) whole cell lysate at 20 µg
Lane 4: Mouse liver tissue lysate at 20 µg
Lane 5: MEF (Mouse embryonic fibroblast (immortalized)) whole cell lysate at 20 µg
Lane 6: Mouse heart tissue lysate at 20 µg
Secondary
All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution
Predicted band size: 21 kDa
Observed band size: 21 kDa
Exposure time: 180s
Immunoprecipitation - Anti-ATF3 antibody [EPR22610-19] - ChIP Grade (ab254268)
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) (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 (Rabbit IgG, monoclonal [EPR25A] - Isotype Control 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.
All lanes: Immunoprecipitation - Anti-ATF3 antibody [EPR22610-19] - ChIP Grade (ab254268)
Predicted band size: 21 kDa
Observed band size: 21 kDa
Western blot - Anti-ATF3 antibody [EPR22610-19] - ChIP Grade (ab254268)
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).
All lanes: Western blot - Anti-ATF3 antibody [EPR22610-19] - ChIP Grade (ab254268) at 1/1000 dilution
Lane 1: HepG2 (human hepatocellular carcinoma epithelial cell), whole cell lysate at 20 µg
Lane 2: HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate at 20 µg
Lane 3: HEK-293T (human embryonic kidney epithelial cell), whole cell lysate at 20 µg
Lane 4: Daudi (human Burkitts lymphoma lymphoblast), whole cell lysate at 20 µg
Lane 5: Wild-type HAP1 whole cell lysate at 20 µg
Lane 6: ATF3 knockout HAP1 whole cell lysate at 20 µg
Secondary
All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution
Predicted band size: 21 kDa
Observed band size: 21 kDa
Exposure time: 26s
Western blot - Anti-ATF3 antibody [EPR22610-19] - ChIP Grade (ab254268)
Blocking and dilution buffer: 5% NFDM/TBST.
The expression profile observed is consistent with what has been described in the literature (PMID: 24973221; 24062788).
All lanes: Western blot - Anti-ATF3 antibody [EPR22610-19] - ChIP Grade (ab254268) at 1/1000 dilution
Lane 1: Untreated THP-1 (human monocytic leukemia monocyte), whole cell lysate at 20 µg
Lane 2: THP-1 treated with 80nM TPA overnight, then treated with 1ug/ml lipopolysaccharide (LPS) for 8 hours, whole cell lysate at 20 µg
Lane 3: Untreated RAW264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage), whole cell lysate at 20 µg
Lane 4: RAW264.7 treated with 1ug/ml lipopolysaccharide (LPS) for 2 hours, whole cell lysate at 20 µg
Secondary
All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution
Predicted band size: 21 kDa
Observed band size: 21 kDa
Exposure time: 70s
Flow Cytometry (Intracellular) - Anti-ATF3 antibody [EPR22610-19] - ChIP Grade (ab254268)
Intracellular 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 (Rabbit IgG, monoclonal [EPR25A] - Isotype Control 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, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) at 1/2000 dilution was used as the secondary antibody.
Immunocytochemistry/ Immunofluorescence - Anti-ATF3 antibody [EPR22610-19] - ChIP Grade (ab254268)
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 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) 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. Alexa Fluor® 594 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker 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 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077 AlexaFluor^® 488 Goat anti-Rabbit secondary at 1/1000 (2 ug/ml) dilution.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ATF3 antibody [EPR22610-19] - ChIP Grade (ab254268)
Immunohistochemical analysis of paraffin-embedded Human 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 Antigen Retrieval Buffer (100X Tris-EDTA Buffer, pH 9.0) 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).
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ATF3 antibody [EPR22610-19] - ChIP Grade (ab254268)
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 Antigen Retrieval Buffer (100X Tris-EDTA Buffer, pH 9.0) 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).
ChIC/CUT&RUN sequencing - Anti-ATF3 antibody [EPR22610-19] - ChIP Grade (ab254268)
ChIC/CUT&RUN was performed using a pAG-MNAse at a final concentration of 700 ng/mL. 2.5X10^5 of Human ATF3 knockout HeLa cell line (Human ATF3 knockout HeLa cell line ab264908) or Human wild-type HeLa cell line (ab255448) were used along with 5µg of Anti-ATF3 antibody (ab254268). Assay Quality Control was conducted using 5µg Anti-CTCF (Anti-CTCF antibody [EPR18253] - ChIP Grade ab188408) on the same cell lines. The resulting DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 10 million reads. The negative IgG control Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730 is also shown.
Additional screenshots of mapped reads can be found in the Protocol booklet in the Support and downloads section. The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.