Anti-ATF3 抗体 [EPR22610-19] - ChIP Grade (ab254268)
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 - ChIP Grade
- Suitable for: ChIC/CUT&RUN-seq, Flow Cyt (Intra), WB, IHC-P, ICC/IF, IP, ChIP
- Knockout validated
- Reacts with: Mouse, Human
Related conjugates and formulations
製品の概要
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製品名
Anti-ATF3 antibody [EPR22610-19] - ChIP Grade
ATF3 一次抗体 製品一覧 -
製品の詳細
Rabbit monoclonal [EPR22610-19] to ATF3 - ChIP Grade -
由来種
Rabbit -
特異性
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.
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アプリケーション
適用あり: ChIC/CUT&RUN-seq, Flow Cyt (Intra), WB, IHC-P, ICC/IF, IP, ChIPmore details -
種交差性
交差種: Mouse, Human -
免疫原
Recombinant fragment. This information is proprietary to Abcam and/or its suppliers.
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ポジティブ・コントロール
- 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; HepG2, HAP1, A549 and HCT116 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.
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特記事項
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
Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.
製品の特性
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製品の状態
Liquid -
保存方法
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. -
バッファー
pH: 7.2
Preservative: 0.01% Sodium azide
Constituents: PBS, 40% Glycerol, 0.05% BSA -
Concentration information loading...
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精製度
Protein A purified -
ポリ/モノ
モノクローナル -
クローン名
EPR22610-19 -
アイソタイプ
IgG -
研究分野
関連製品
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Alternative Versions
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Compatible Secondaries
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Isotype control
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KO cell lines
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KO cell lysates
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Related Products
アプリケーション
The Abpromise guarantee
Abpromise保証は、 次のテスト済みアプリケーションにおけるab254268の使用に適用されます
アプリケーションノートには、推奨の開始希釈率がありますが、適切な希釈率につきましてはご検討ください。
アプリケーション | Abreviews | 特記事項 |
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ChIC/CUT&RUN-seq |
Use at an assay dependent concentration.
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Flow Cyt (Intra) |
1/600.
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WB |
1/1000. Detects a band of approximately 21 kDa (predicted molecular weight: 21 kDa).
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IHC-P |
1/500. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
IHC is not recommended for mouse. |
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ICC/IF |
1/100.
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IP |
1/30.
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ChIP |
Use 5 µg for 25 µg of chromatin.
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特記事項 |
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ChIC/CUT&RUN-seq
Use at an assay dependent concentration. |
Flow Cyt (Intra)
1/600. |
WB
1/1000. Detects a band of approximately 21 kDa (predicted molecular weight: 21 kDa). |
IHC-P
1/500. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol. IHC is not recommended for mouse. |
ICC/IF
1/100. |
IP
1/30. |
ChIP
Use 5 µg for 25 µg of chromatin. |
ターゲット情報
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機能
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. -
配列類似性
Belongs to the bZIP family. ATF subfamily.
Contains 1 bZIP domain. -
細胞内局在
Nucleus. - Information by UniProt
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参照データベース
- Entrez Gene: 467 Human
- Entrez Gene: 11910 Mouse
- Omim: 603148 Human
- SwissProt: P18847 Human
- SwissProt: Q60765 Mouse
- Unigene: 460 Human
- Unigene: 2706 Mouse
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別名
- Activating transcription factor 3 antibody
- ATF3 antibody
- ATF3_HUMAN antibody
see all
画像
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ChIC/CUT&RUN was performed using a pAG-MNAse at a final concentration of 700 ng/µL. 2.5X10^5 of 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 (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 ab172730 is also shown.
Additional screenshots of mapped reads can be downloaded here. The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods. -
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 kDaLanes 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.
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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 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) (ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to mouse IgG (Alexa Fluor® 594) (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).
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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 kDaLanes 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.
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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 secondsBlocking/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).
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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 1/100000 dilution
Predicted band size: 21 kDa
Observed band size: 21 kDa
Exposure time: 26 secondsBlocking 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).
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All lanes : Anti-ATF3 antibody [EPR22610-19] - ChIP Grade (ab254268) at 1/1000 dilution
Lane 1 : Untreated THP-1 (human monocytic leukemia monocyte), whole cell lysate
Lane 2 : THP-1 treated with 80nM TPA overnight, then treated with 1ug/ml lipopolysaccharide (LPS) for 8 hours, whole cell lysate
Lane 3 : Untreated RAW264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage), whole cell lysate
Lane 4 : RAW264.7 treated with 1ug/ml lipopolysaccharide (LPS) for 2 hours, whole cell lysate
Lysates/proteins at 20 µg per lane.
Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution
Predicted band size: 21 kDa
Observed band size: 21 kDa
Exposure time: 70 secondsBlocking and dilution buffer: 5% NFDM/TBST.
The expression profile observed is consistent with what has been described in the literature (PMID: 24973221; 24062788).
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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.
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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.
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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 (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.
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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.
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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 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).
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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).
データシートおよび資料
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SDS download
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Datasheet download
Certificate of Compliance
参考文献 (13)
ab254268 は 13 報の論文で使用されています。
- Cheng Z et al. Activating transcription factor 3-activated long noncoding RNA forkhead box P4-antisense RNA 1 aggravates colorectal cancer progression by regulating microRNA-423-5p/nucleus accumbens associated 1 axis. Bioengineered 13:2114-2129 (2022). PubMed: 35034547
- Smargon AA et al. Crosstalk between CRISPR-Cas9 and the human transcriptome. Nat Commun 13:1125 (2022). PubMed: 35236841
- Wu YL et al. Salvia miltiorrhiza Extract and Individual Synthesized Component Derivatives Induce Activating-Transcription-Factor-3-Mediated Anti-Obesity Effects and Attenuate Obesity-Induced Metabolic Disorder by Suppressing C/EBPα in High-Fat-Induced Obese Mice. Cells 11:N/A (2022). PubMed: 35326476
- Wei R et al. Screening and Identification of Hub Genes in the Development of Early Diabetic Kidney Disease Based on Weighted Gene Co-Expression Network Analysis. Front Endocrinol (Lausanne) 13:883658 (2022). PubMed: 35721731
- Guo W et al. Nanoparticle delivery of miR-21-3p sensitizes melanoma to anti-PD-1 immunotherapy by promoting ferroptosis. J Immunother Cancer 10:N/A (2022). PubMed: 35738798