重组Anti-GCN2抗体[EPR5970(2)] (ab134053)
Key features and details
- Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
- Rabbit monoclonal [EPR5970(2)] to GCN2
- Suitable for: WB, IHC-P, Flow Cyt (Intra), ICC/IF
- Knockout validated
- Reacts with: Human
Related conjugates and formulations
概述
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产品名称
Anti-GCN2抗体[EPR5970(2)]
参阅全部 GCN2 一抗 -
描述
兔单克隆抗体[EPR5970(2)] to GCN2 -
宿主
Rabbit -
特异性
This antibody does not react with mouse and rat species in Western blot application.
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经测试应用
适用于: WB, IHC-P, Flow Cyt (Intra), ICC/IFmore details
不适用于: IP -
种属反应性
与反应: Human -
免疫原
Synthetic peptide corresponding to Human GCN2 (N terminal).
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阳性对照
- WB: HEK-293T, HAP1, HeLa, 293T, MOLT4, MCF7 and A549 cell lysates. ICC/IF: MCF7 cells. Flow Cyt (intra): MCF7 and HeLa cells. IHC-P: Human kidney and human breast carcinoma tissue.
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常规说明
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.
Mouse, Rat: We have preliminary internal testing data to indicate this antibody may not react with these species. Please contact us for more information.
性能
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形式
Liquid -
存放说明
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. Stable for 12 months at -20°C. -
存储溶液
pH: 7.20
Preservative: 0.01% Sodium azide
Constituents: 0.05% BSA, 40% Glycerol, 59% PBS -
Concentration information loading...
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纯度
Protein A purified -
克隆
单克隆 -
克隆编号
EPR5970(2) -
同种型
IgG -
研究领域
相关产品
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Alternative Versions
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Compatible Secondaries
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Conjugation kits
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Isotype control
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KO cell lines
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KO cell lysates
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Positive Controls
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Recombinant Protein
应用
The Abpromise guarantee
Abpromise™承诺保证使用ab134053于以下的经测试应用
“应用说明”部分 下显示的仅为推荐的起始稀释度;实际最佳的稀释度/浓度应由使用者检定。
应用 | Ab评论 | 说明 |
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WB | (2) |
1/1000 - 1/10000. Detects a band of approximately 220 kDa (predicted molecular weight: 187 kDa).
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IHC-P |
1/100 - 1/250. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
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Flow Cyt (Intra) |
1/100.
ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody. For unpurified use at 1/1000 - 1/10000 |
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ICC/IF |
1/250 - 1/500.
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说明 |
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WB
1/1000 - 1/10000. Detects a band of approximately 220 kDa (predicted molecular weight: 187 kDa). |
IHC-P
1/100 - 1/250. Perform heat mediated antigen retrieval before commencing with IHC staining protocol. |
Flow Cyt (Intra)
1/100. ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody. For unpurified use at 1/1000 - 1/10000 |
ICC/IF
1/250 - 1/500. |
靶标
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功能
Can phosphorylate the alpha subunit of EIF2 and may mediate translational control. -
组织特异性
Widely expressed. -
序列相似性
Belongs to the protein kinase superfamily. Ser/Thr protein kinase family. GCN2 subfamily.
Contains 2 protein kinase domains.
Contains 1 RWD domain. -
结构域
Kinase domain 1 is a degenerate kinase domain.
RWD domain is reported to interact with GCN1L1. -
翻译后修饰
Autophosphorylated on threonine residues. - Information by UniProt
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数据库链接
- Entrez Gene: 440275 Human
- Omim: 609280 Human
- SwissProt: Q9P2K8 Human
- Unigene: 656673 Human
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别名
- E2AK4_HUMAN antibody
- Eif2ak4 antibody
- Eukaryotic Translation Initiation Factor 2 alpha kinase 4 antibody
see all
图片
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All lanes : Anti-GCN2 antibody [EPR5970(2)] (ab134053) at 1/1000 dilution
Lane 1 : Wild-type HEK-293T cell lysate
Lane 2 : EIF2AK4 knockout HEK-293T cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 187 kDa
Observed band size: 187 kDaLanes 1- 2: Merged signal (red and green). Green - ab134053 observed at 187 kDa. Red - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) observed at 50 kDa.
ab134053 was shown to react with GCN2 in wild-type HEK-293T cells in western blot. Loss of signal was observed when knockout cell line ab267247 (knockout cell lysate ab256903) was used. Wild-type HEK-293T and EIF2AK4 knockout HEK-293T cell lysates were subjected to SDS-PAGE. ab134053 and Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) overnight at 4°C at a 1 in 1000 dilution and a 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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Immunocytochemistry/ Immunofluorescence analysis of MCF7 (Human breast adenocarcinoma cell line) cells labeling GCN2 with ab134053 at 1/250 dilution (4.0μg/ml). The cells were co-stained with ab195889, an Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) at 1/200 (2.5 μg/ml). Cells were fixed with 100% methanol. ab150077, a Goat anti-rabbit IgG(Alexa Fluor® 488) secondary antibody was used at 1/1000 dilution. DAPI was used as the nuclear counter stain.
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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GCN2 antibody [EPR5970(2)] (ab134053)
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human breast carconoma tissue sections labeling GCN2 with purified ab134053 at 1/100 dilution (10 μg/ml). Heat mediated antigen retrieval was performed using EDTA Buffer, PH9. ab97051, a Goat Anti-Rabbit IgG H&L (HRP) secondary antibody was used at 1/500 dilution. Tissue was counterstained with hematoxylin. PBS instead of the primary antibody was used as the negative control.
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Intracellular Flow Cytometry analysis of MCF7 (Human breast adenocarcinoma cell line) cells labeling GCN2 with purified ab134053 at 1/100 dilution (10 ug/ml). Cells were fixed with 4% paraformaldehyde. A Goat anti-rabbit IgG (Alexa Fluor® 488) secondary antibody was used at 1/2000 dilution. Rabbit monoclonal IgG (Black) was used as the isotypre control. Cells without incubation with the primary antibody and secondary antibody (Blue) is the unlabeled control.
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All lanes : Anti-GCN2 antibody [EPR5970(2)] (ab134053) at 1/1000 dilution
Lane 1 : Wild-type HEK-293T cell lysate
Lane 2 : EIF2AK4 knockout HEK-293T cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 187 kDa
Observed band size: 187 kDaLanes 1- 2: Merged signal (red and green). Green - ab134053 observed at 187 kDa. Red - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) observed at 50 kDa.
ab134053 was shown to react with GCN2 in wild-type HEK-293T cells in western blot. Loss of signal was observed when knockout cell line ab267246 (knockout cell lysate ab256902) was used. Wild-type HEK-293T and EIF2AK4 knockout HEK-293T cell lysates were subjected to SDS-PAGE. ab134053 and Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) overnight at 4°C at a 1 in 1000 Dilution and a 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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Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: GCN2 knockout HAP1 cell lysate (20 µg)
Lane 3: MOLT4 cell lysate (20 µg)
Lane 4: A549 cell lysate (20 µg)
Lanes 1 - 4: Merged signal (red and green). Green - ab134053 observed at 171 kDa. Red - loading control, ab18058, observed at 124 kDa.Unpurified ab134053 was shown to recognize GCN2 when GCN2 knockout samples were used, along with additional cross-reactive bands. Wild-type and GCN2 knockout samples were subjected to SDS-PAGE. ab134053 and ab18058 (loading control to Vinculin) were diluted at 1/1000 and 1/10 000 respectively and incubated overnight at 4°C. 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 1/10000 dilution for 1 h at room temperature before imaging.
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All lanes : Anti-GCN2 antibody [EPR5970(2)] (ab134053) at 1/10000 dilution (purified)
Lane 1 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 2 : HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate
Lysates/proteins at 15 µg per lane.
Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution
Predicted band size: 187 kDa
Observed band size: 187 kDaBlocking and diluting buffer: 5% NFDM/TBST.
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Anti-GCN2 antibody [EPR5970(2)] (ab134053) at 1/50000 dilution (purified) + MOLT-4 (Human lymphoblastic leukemia cell line) whole cell lysate at 20 µg
Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution
Predicted band size: 187 kDa
Observed band size: 187 kDaBlocking and diluting buffer: 5% NFDM /TBST.
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All lanes : Anti-GCN2 antibody [EPR5970(2)] (ab134053) at 1/1000 dilution (Unpurified)
Lane 1 : HeLa cell lysate
Lane 2 : 293T cell lysate
Lane 3 : MOLT4 cell lysate
Lane 4 : A549 cell lysate
Lysates/proteins at 10 µg per lane.
Secondary
All lanes : HRP labelled goat anti-rabbit at 1/2000 dilution
Predicted band size: 187 kDa -
Immunofluorescence staining of MCF-7 cells with purified ab134053 at a working dilution of 1/500, counter-stained with DAPI. The secondary antibody was an Alexa Fluor® 488 conjugated goat anti-rabbit (ab150077), used at a dilution of 1/1000. The cells were fixed in 100% methanol. The negative control is shown in bottom right hand panel - for the negative control, PBS was used instead of the primary antibody.
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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GCN2 antibody [EPR5970(2)] (ab134053)
Immunohistochemical analysis of paraffin-embedded Human kidney tissue labelling GCN2 with unpurified ab134053 at 1/100 dilution.
Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
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Overlay histogram showing HeLa cells stained with unpurified ab134053 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab134053, 1/10000 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor® 488 goat anti-rabbit IgG (H&L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (0.1µg/1x106 cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter. This antibody gave a positive signal in HeLa cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.
实验方案
数据表及文件
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SDS download
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Datasheet download
文献 (12)
ab134053 被引用在 12 文献中.
- Takahashi M et al. Activating mutations in EGFR and PI3K promote ATF4 induction for NSCLC cell survival during amino acid deprivation. Heliyon 9:e14799 (2023). PubMed: 37025861
- Tang CP et al. GCN2 kinase activation by ATP-competitive kinase inhibitors. Nat Chem Biol 18:207-215 (2022). PubMed: 34949839
- Missiaen R et al. GCN2 inhibition sensitizes arginine-deprived hepatocellular carcinoma cells to senolytic treatment. Cell Metab 34:1151-1167.e7 (2022). PubMed: 35839757
- Datta C et al. Ribosome changes reprogram translation for chemosurvival in G0 leukemic cells. Sci Adv 8:eabo1304 (2022). PubMed: 36306353
- Ma B et al. Patient-specific and gene-corrected induced pluripotent stem cell-derived endothelial cells elucidate single-cell phenotype of pulmonary veno-occlusive disease. Stem Cell Reports 17:2674-2689 (2022). PubMed: 36400028