Anti-EGFR 抗体 [E234] (ab32198)
Key features and details
- Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
- Rabbit monoclonal [E234] to EGFR
- Suitable for: Flow Cyt (Intra), WB, ICC/IF, IHC-P
- Knockout validated
- Reacts with: Human
Related conjugates and formulations
製品の概要
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製品名
Anti-EGFR antibody [E234]
EGFR 一次抗体 製品一覧 -
製品の詳細
Rabbit monoclonal [E234] to EGFR -
由来種
Rabbit -
特異性
b32198 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.
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アプリケーション
適用あり: Flow Cyt (Intra), WB, ICC/IF, IHC-Pmore details -
種交差性
交差種: Human
交差が予測される動物種: Cow, Pig -
免疫原
Synthetic peptide corresponding to Human EGFR aa 1-100 (N terminal).
Database link: P00533 -
ポジティブ・コントロール
- WB: HeLa, A549 and A431 cell lysates IHC-P: FFPE human skin tissue sections, human pancreatic carcinoma tissue. ICC/IF: A431 cells. Flow Cyt (intra): A431 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.
Mouse, Rat: We have preliminary internal testing data to indicate this antibody may not react with these species. Please contact us for more information.
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: 59% 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
E234 -
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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Isotype control
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KO cell lines
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Positive Controls
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Recombinant Protein
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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ab32198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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Flow Cyt (Intra) |
1/200.
For unpurified use at 1/50. ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody. |
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WB |
1/1000. Detects a band of approximately 134 kDa (predicted molecular weight: 180 kDa).
For unpurified use at 1/500. 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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ICC/IF | (1) |
1/100.
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IHC-P | (1) |
1/100. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
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特記事項 |
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Flow Cyt (Intra)
1/200. For unpurified use at 1/50. ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody. |
WB
1/1000. Detects a band of approximately 134 kDa (predicted molecular weight: 180 kDa). For unpurified use at 1/500. 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. |
ICC/IF
1/100. |
IHC-P
1/100. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol. |
ターゲット情報
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機能
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. -
組織特異性
Ubiquitously expressed. Isoform 2 is also expressed in ovarian cancers. -
関連疾患
Lung cancer
Inflammatory skin and bowel disease, neonatal, 2 -
配列類似性
Belongs to the protein kinase superfamily. Tyr protein kinase family. EGF receptor subfamily.
Contains 1 protein kinase domain. -
翻訳後修飾
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. -
細胞内局在
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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参照データベース
- Entrez Gene: 407217 Cow
- Entrez Gene: 1956 Human
- Entrez Gene: 397070 Pig
- Omim: 131550 Human
- SwissProt: P00533 Human
- Unigene: 488293 Human
- Unigene: 605083 Human
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別名
- Avian erythroblastic leukemia viral (v erb b) oncogene homolog antibody
- Cell growth inhibiting protein 40 antibody
- Cell proliferation inducing protein 61 antibody
see all
画像
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All lanes : Anti-EGFR antibody [E234] (ab32198) at 1/1000 dilution
Lane 1 : Wild-type A549 cell lysate
Lane 2 : EGFR knockout A549 cell lysate
Lane 3 : Wild-type HeLa cell lysate
Lane 4 : EGFR knockout HeLa cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 180 kDa
Observed band size: 160 kDa why is the actual band size different from the predicted?Western blot: Anti-EGFR antibody [E234] (ab32198) staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab32198 was shown to bind specifically to EGFR. A band was observed at 160 kDa in wild-type cell lysates with no signal observed at this size in EGFR knockout cell lines. To generate this image, wild-type and EGFR knockout A549 (ab286394) and HeLa (ab255385) cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3% milk in TBS-0.1% Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4°C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.
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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human pancreatic carcinoma tissue sections labeling EGFR with purified ab32198 at 1/100 dilution (2.22 µg/mL). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). ImmunoHistoProbe one step HRP Polymer (ready to use) was used as the secondary antibody. Negative control: PBS instead of the primary antibody. Hematoxylin was used as a counterstain.
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All lanes : Anti-EGFR antibody [E234] (ab32198) at 1/1000 dilution (Purified)
Lane 1 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates
Lane 2 : A431 (Human epidermoid carcinoma epithelial cell) whole cell lysates
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: 180 kDa
Observed band size: 150 kDa why is the actual band size different from the predicted?Blocking/Diluting Buffer and concentration: 5% NFDM/TBST
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Immunocytochemistry/ Immunofluorescence analysis of A431 (Human epidermoid carcinoma epithelial cell) cells labeling EGFR with purified ab32198 at 1/100 dilution (2.2 µg/mL). Cells were fixed in 100% Methanol. Cells were counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) 1/200 (2.5 µg/mL). Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) was used as the secondary antibody at 1/1000 (2 µg/mL) dilution. DAPI (blue) was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.
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Intracellular Flow Cytometry analysis of A431 (Human epidermoid carcinoma epithelial cell) cells labeling EGFR with purified ab32198 at 1/200 dilution (10µg/mL) (Red). Cells were fixed with 80% Methanol and permeabilised with 0.1% Tween-20. A Goat anti rabbit IgG (Alexa Fluorr® 488, ab150077) secondary antibody was used at 1/2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).
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ab32198 (unpurified) stained A431 cells. The cells were 100% methanol fixed for 10 minutes at -20°C and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1hour at room temperature to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab32198 at 1in100 dilution) overnight at +4°C. The secondary antibody (pseudo-colored green) was Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed (ab150081) used at a 1/1000 dilution for 1hour at room temperature. Alexa Fluor® 594 WGA was used to label plasma membranes (pseudo-colored red) at a 1/200 dilution for 1hour at room temperature. DAPI was used to stain the cell nuclei (pseudo-colored blue) at a concentration of 1.43µM for 1hour at room temperature.
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IHC image of EGFR staining in human skin formalin fixed paraffin embedded tissue section*, performed on a Leica Bond™ system using the standard protocol F. 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 ab32198 (unpurified) at 1/100 dilution for 15 mins at room temperature and detected using an HRP conjugated compact polymer 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.
*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre
プロトコール
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.
データシートおよび資料
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SDS download
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Datasheet download
参考文献 (4)
ab32198 は 4 報の論文で使用されています。
- Governa V et al. Landscape of surfaceome and endocytome in human glioma is divergent and depends on cellular spatial organization. Proc Natl Acad Sci U S A 119:N/A (2022). PubMed: 35217608
- Paterson K et al. Assessment of CAR-T Cell-Mediated Cytotoxicity in 3D Microfluidic Cancer Co-Culture Models for Combination Therapy. IEEE Open J Eng Med Biol 3:86-95 (2022). PubMed: 35813488
- Tan D et al. Screening of an individualized treatment strategy for an advanced gallbladder cancer using patient-derived tumor xenograft and organoid models. Front Oncol 12:1043479 (2022). PubMed: 36591461
- Gireud-Goss M et al. Distinct mechanisms enable inward or outward budding from late endosomes/multivesicular bodies. Exp Cell Res 372:1-15 (2018). PubMed: 30144444