重组Anti-EGFR抗体[E235] (ab32077)
Key features and details
- Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
- Rabbit monoclonal [E235] to EGFR
- Suitable for: IHC-P, ICC/IF, WB, Flow Cyt, IP
- Knockout validated
- Reacts with: Mouse, Rat, Human
概述
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产品名称
Anti-EGFR抗体[E235]
参阅全部 EGFR 一抗 -
描述
兔单克隆抗体[E235] to EGFR -
宿主
Rabbit -
特异性
ab32077 detects Epidermal growth factor receptor (EGFR). It does not cross react with other ERBB family members. This product yielded a strong signal in western blot using A431 (human squamous carcinoma) lysate which naturally overexpresses the EGFR protein. Western blot conditions may need to be optimised for cell lines and tissues that express lower levels of endogenous EGFR. -
Tested Applications & Species
Application Species Flow Cyt HumanICC/IF HumanIHC-P MouseIP HumanWB Human -
免疫原
Synthetic peptide within Human EGFR aa 1150 to the C-terminus (C terminal). The exact sequence is proprietary.
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阳性对照
- WB: A431 cell lysate. ICC/IF: A431 cells. Flow Cyt: A431 cells. IHC-P: FFPE mouse skin normal. IP: A431 cell lysate
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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.
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.
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. Avoid freeze / thaw cycle. -
存储溶液
pH: 7.20
Preservative: 0.01% Sodium azide
Constituents: 49% PBS, 50% Glycerol (glycerin, glycerine), 0.05% BSA -
Concentration information loading...
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纯度
Protein A purified -
克隆
单克隆 -
克隆编号
E235 -
同种型
IgG -
研究领域
相关产品
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Affibody® Molecule
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Alternative Versions
- Alexa Fluor® 488 Anti-EGFR antibody [E235] (ab205731)
- Alexa Fluor® 647 Anti-EGFR antibody [E235] (ab205732)
- HRP Anti-EGFR antibody [E235] (ab206036)
- PE Anti-EGFR antibody [E235] (ab213332)
- Alexa Fluor® 555 Anti-EGFR antibody [E235] (ab214627)
- Anti-EGFR antibody [E235] - BSA and Azide free (ab227459)
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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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KO cell pellets
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Positive Controls
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Related Products
应用
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab32077 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Tested applications are guaranteed to work and covered by our Abpromise guarantee.
Predicted to work for this combination of applications and species but not guaranteed.
Does not work for this combination of applications and species.
应用 | Species |
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Flow Cyt |
Human
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ICC/IF |
Human
|
IHC-P |
Mouse
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IP |
Human
|
WB |
Human
|
应用 | Ab评论 | 说明 |
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IHC-P | (3) |
1/100 - 1/200. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
|
ICC/IF |
1/500.
|
|
WB |
1/1000 - 1/10000. Detects a band of approximately 180 kDa (predicted molecular weight: 134 kDa).
This product yielded a strong signal in western blot using A431 (human squamous carcinoma) lysate which naturally overexpresses the EGFR protein. Western blot conditions may need to be optimised for cell lines and tissues that express lower levels of endogenous EGFR. |
|
Flow Cyt |
1/10 - 1/1000.
ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody. |
|
IP |
1/50.
|
说明 |
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IHC-P
1/100 - 1/200. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol. |
ICC/IF
1/500. |
WB
1/1000 - 1/10000. Detects a band of approximately 180 kDa (predicted molecular weight: 134 kDa). This product yielded a strong signal in western blot using A431 (human squamous carcinoma) lysate which naturally overexpresses the EGFR protein. Western blot conditions may need to be optimised for cell lines and tissues that express lower levels of endogenous EGFR. |
Flow Cyt
1/10 - 1/1000. ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody. |
IP
1/50. |
靶标
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功能
Receptor tyrosine kinase binding ligands of the EGF family and activating several signaling cascades to convert extracellular cues into appropriate cellular responses. Known ligands include EGF, TGFA/TGF-alpha, amphiregulin, epigen/EPGN, BTC/betacellulin, epiregulin/EREG and HBEGF/heparin-binding EGF. Ligand binding triggers receptor homo- and/or heterodimerization and autophosphorylation on key cytoplasmic residues. The phosphorylated receptor recruits adapter proteins like GRB2 which in turn activates complex downstream signaling cascades. Activates at least 4 major downstream signaling cascades including the RAS-RAF-MEK-ERK, PI3 kinase-AKT, PLCgamma-PKC and STATs modules. May also activate the NF-kappa-B signaling cascade. Also directly phosphorylates other proteins like RGS16, activating its GTPase activity and probably coupling the EGF receptor signaling to the G protein-coupled receptor signaling. Also phosphorylates MUC1 and increases its interaction with SRC and CTNNB1/beta-catenin.
Isoform 2 may act as an antagonist of EGF action. -
组织特异性
Ubiquitously expressed. Isoform 2 is also expressed in ovarian cancers. -
疾病相关
Lung cancer
Inflammatory skin and bowel disease, neonatal, 2 -
序列相似性
Belongs to the protein kinase superfamily. Tyr protein kinase family. EGF receptor subfamily.
Contains 1 protein kinase domain. -
翻译后修饰
Phosphorylation at Ser-695 is partial and occurs only if Thr-693 is phosphorylated. Phosphorylation at Thr-678 and Thr-693 by PRKD1 inhibits EGF-induced MAPK8/JNK1 activation. Dephosphorylation by PTPRJ prevents endocytosis and stabilizes the receptor at the plasma membrane. Autophosphorylation at Tyr-1197 is stimulated by methylation at Arg-1199 and enhances interaction with PTPN6. Autophosphorylation at Tyr-1092 and/or Tyr-1110 recruits STAT3. Dephosphorylated by PTPN1 and PTPN2.
Monoubiquitinated and polyubiquitinated upon EGF stimulation; which does not affect tyrosine kinase activity or signaling capacity but may play a role in lysosomal targeting. Polyubiquitin linkage is mainly through 'Lys-63', but linkage through 'Lys-48', 'Lys-11' and 'Lys-29' also occurs. Deubiquitination by OTUD7B prevents degradation. Ubiquitinated by RNF115 and RNF126.
Methylated. Methylation at Arg-1199 by PRMT5 stimulates phosphorylation at Tyr-1197. -
细胞定位
Secreted and Cell membrane. Endoplasmic reticulum membrane. Golgi apparatus membrane. Nucleus membrane. Endosome. Endosome membrane. Nucleus. In response to EGF, translocated from the cell membrane to the nucleus via Golgi and ER. Endocytosed upon activation by ligand. Colocalized with GPER1 in the nucleus of estrogen agonist-induced cancer-associated fibroblasts (CAF). - Information by UniProt
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数据库链接
- Entrez Gene: 1956 Human
- Entrez Gene: 13649 Mouse
- Entrez Gene: 24329 Rat
- Omim: 131550 Human
- SwissProt: P00533 Human
- SwissProt: Q01279 Mouse
- Unigene: 488293 Human
- Unigene: 605083 Human
see all -
别名
- Avian erythroblastic leukemia viral (v erb b) oncogene homolog antibody
- Cell growth inhibiting protein 40 antibody
- Cell proliferation inducing protein 61 antibody
see all
图片
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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-EGFR antibody [E235] (ab32077)
IHC image of EGFR staining in a formalin fixed, paraffin embedded mouse normal skin tissue section, performed on a Leica Bond™ system using the standard protocol B. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab32077 at 1/200 dilution for 15 mins at room temperature. A goat anti-rabbit biotinylated secondary antibody was used to detect the primary, and visualized using an HRP conjugated ABC system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.
For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.
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All lanes : Anti-EGFR antibody [E235] (ab32077) at 1/1000 dilution
Lane 1 : Caco-2 cell lysate
Lane 2 : A431 cell lysate
Lane 3 : Mouse skin cell lysate
Lane 4 : Rat skin cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 134 kDaThis blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour before being incubated with ab32077 overnight at 4°C in the presence of loading control ab18058 (Mouse monoclonal [SPM227] to Vinculin diluted 1:10000). Antibody binding was detected using IR-labelled goat anti-Rabbit Ab at a 1:10,000 dilution for one hour at room temperature before imaging.
This product yielded a strong signal in western blot using A431 (human squamous carcinoma) lysate which naturally overexpresses the EGFR protein. Western blot conditions may need to be optimised for cell lines and tissues that express lower levels of endogenous EGFR.
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Purified ab32077 at 1/50 dilution (2µg) immunoprecipitating EGFR in A431 whole cell lysate.
Lane 1 (input): A431 (Human epidermoid carcinoma epithelial cell) whole cell lysate 10µg
Lane 2 (+): ab32077 + A431 whole cell lysate.
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab32077 in A431 whole cell lysate.
VeriBlot for IP Detection Reagent (HRP) (ab131366) (1/1000 dilution) was used for Western blotting.
Blocking Buffer and concentration: 5% NFDM/TBST.
Diluting buffer and concentration: 5% NFDM/TBST.
Observed band size: 180 kDa -
All lanes : Anti-EGFR antibody [E235] (ab32077)
Lane 1 : A431 cell lysate
Lane 2 : MDA-MB-468 cell lysate
Lane 3 : Wild-type HeLa cell lysate
Lane 4 : EGFR knockout HeLa cell lysate
Lysates/proteins at 20 µg per lane.
Predicted band size: 134 kDaLanes 1 - 4: Merged signal (red and green). Green - ab32077 observed at 175 kDa. Red - loading control, ab130007 observed at 125 kDa.
ab32077 was shown to react with EGFR in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab255385 (knockout cell lysate ab263845) was used. Wild-type and EGFR knockout samples were subjected to SDS-PAGE. ab32077 and Anti-Vinculin antibody [VIN-54] (ab130007) were incubated overnight at 4°C at 1 in 1000 dilution 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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Immunocytochemistry/Immunofluorescence analysis of A431 cells labelling EGFR with ab32077 at 1/500. Cells were fixed with 100% methanol. ab150077, an Alexa Fluor® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody.
Control: PBS only.
Nuclear counter stain: DAPI. -
Overlay histogram showing A431 cells stained with ab32077 (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 (ab32077, 1/1000 dilution) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 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.
实验方案
To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.
数据表及文件
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SDS download
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Datasheet download
文献 (10)
ab32077 被引用在 10 文献中.
- Ayanlaja AA et al. Doublecortin undergo nucleocytoplasmic transport via the RanGTPase signaling to promote glioma progression. Cell Commun Signal 18:24 (2020). PubMed: 32050972
- Nagpal A et al. Neoadjuvant neratinib promotes ferroptosis and inhibits brain metastasis in a novel syngeneic model of spontaneous HER2+ve breast cancer metastasis. Breast Cancer Res 21:94 (2019). PubMed: 31409375
- Spence JS et al. IFITM3 directly engages and shuttles incoming virus particles to lysosomes. Nat Chem Biol 15:259-268 (2019). PubMed: 30643282
- Shao B et al. The 3p14.2 tumour suppressor ADAMTS9 is inactivated by promoter CpG methylation and inhibits tumour cell growth in breast cancer. J Cell Mol Med 22:1257-1271 (2018). WB ; Human . PubMed: 29193730
- Mansour AM et al. Epidermal growth factor expression as a predictor of chemotherapeutic resistance in muscle-invasive bladder cancer. BMC Urol 18:100 (2018). PubMed: 30413194
- Randall EC et al. Integrated mapping of pharmacokinetics and pharmacodynamics in a patient-derived xenograft model of glioblastoma. Nat Commun 9:4904 (2018). PubMed: 30464169
- Tian C et al. ABCG1 as a potential oncogene in lung cancer. Exp Ther Med 13:3189-3194 (2017). PubMed: 28588672
- Gao L et al. Efficient Generation of Mice with Consistent Transgene Expression by FEEST. Sci Rep 5:16284 (2015). PubMed: 26573149
- Xu X et al. Establishment and characterization of GCSR1, a multi-drug resistant signet ring cell gastric cancer cell line. Int J Oncol 46:2479-87 (2015). PubMed: 25892440
- Hofmann K et al. A novel alkyne cholesterol to trace cellular cholesterol metabolism and localization. J Lipid Res 55:583-91 (2014). PubMed: 24334219