Anti-EGFR 抗体 [EP38Y] (ab52894)
Key features and details
- Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
- Rabbit monoclonal [EP38Y] to EGFR
- Suitable for: WB, IP, Flow Cyt, IHC-P, ICC/IF
- Knockout validated
- Reacts with: Mouse, Rat, Human
製品の概要
-
製品名
Anti-EGFR antibody [EP38Y]
EGFR 一次抗体 製品一覧 -
製品の詳細
Rabbit monoclonal [EP38Y] to EGFR -
由来種
Rabbit -
特異性
The immunogen for this product is a synthetic phospho-peptide corresponding to residues surrounding Tyr1068 of human EGFR. After screening, clone “EP38Y” was found to recognize total EGFR and is not specific to phosphorylated-Tyr1068 EGFR. 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. The mouse and rat recommendation is based on the WB results. This antibody may not be suitable for IHC with mouse or rat samples. -
アプリケーション
適用あり: WB, IP, Flow Cyt, IHC-P, ICC/IFmore details -
種交差性
交差種: Mouse, Rat, Human -
免疫原
-
ポジティブ・コントロール
- ICC/IF: A431 cells. WB: HeLa, Caco-2 and A431 cell lysate; Rat liver and skin lysate; Mouse lung and skin lysate IP: HeLa whole cell lysate (ab150035). Flow Cyt: A431 cells. IHC-P: Human cervical carcinoma
-
特記事項
Abcam recommended secondaries - Goat Anti-Rabbit HRP (ab205718) and Goat Anti-Rabbit Alexa Fluor® 488 (ab150077).
See other anti-rabbit secondary antibodies that can be used with this antibody.
Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.
We are constantly working hard to ensure we provide our customers with best in class antibodies. As a result of this work we are pleased to now offer this antibody in purified format. We are in the process of updating our datasheets. The purified format is designated 'PUR' on our product labels. If you have any questions regarding this update, please contact our Scientific Support team.
Reproducibility is key to advancing scientific discovery and accelerating scientists’ next breakthrough.
Abcam is leading the way with our range of recombinant antibodies, knockout-validated antibodies and knockout cell lines, all of which support improved reproducibility.
We are also planning to innovate the way in which we present recommended applications and species on our product datasheets, so that only applications & species that have been tested in our own labs, our suppliers or by selected trusted collaborators are covered by our Abpromise™ guarantee.
In preparation for this, we have started to update the applications & species that this product is Abpromise guaranteed for.
We are also updating the applications & species that this product has been “predicted to work with,” however this information is not covered by our Abpromise guarantee.
Applications & species from publications and Abreviews that have not been tested in our own labs or in those of our suppliers are not covered by the Abpromise guarantee.
Please check that this product meets your needs before purchasing. If you have any questions, special requirements or concerns, please send us an inquiry and/or contact our Support team ahead of purchase. Recommended alternatives for this product can be found below, as well as customer reviews and Q&As.
製品の特性
-
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. Avoid freeze / thaw cycle. -
解離定数(KD 値)
KD = 1.90 x 10 -11 M Learn more about KD -
バッファー
pH: 7.20
Preservative: 0.01% Sodium azide
Constituents: PBS, 59% Glycerol (glycerin, glycerine), 0.05% BSA -
Concentration information loading...
-
精製度
Protein A purified -
ポリ/モノ
モノクローナル -
クローン名
EP38Y -
アイソタイプ
IgG -
研究分野
関連製品
-
Alternative Versions
- Anti-EGFR antibody [EP38Y] - Low endotoxin, Azide free (ab174481)
- Alexa Fluor® 647 Anti-EGFR antibody [EP38Y] (ab192982)
- Alexa Fluor® 488 Anti-EGFR antibody [EP38Y] (ab193244)
- HRP Anti-EGFR antibody [EP38Y] (ab193602)
- Alexa Fluor® 594 Anti-EGFR antibody [EP38Y] (ab207870)
- PE Anti-EGFR antibody [EP38Y] (ab208753)
- Anti-EGFR antibody [EP38Y] - BSA and Azide free (ab272293)
- Alexa Fluor® 555 Anti-EGFR antibody [EP38Y] (ab274892)
-
Compatible Secondaries
-
Immunizing Peptide (Blocking)
-
Isotype control
-
KO cell lines
-
KO cell lysates
-
Positive Controls
-
Recombinant Protein
-
Related Products
アプリケーション
Our Abpromise guarantee covers the use of ab52894 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
アプリケーション | Abreviews | 特記事項 |
---|---|---|
WB | 1/1000 - 1/10000. Detects a band of approximately 175 kDa (predicted molecular weight: 134 kDa).Can be blocked with EGFR peptide (ab204282). 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 |
|
IP | 1/20. | |
Flow Cyt | 1/20. ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody. |
|
IHC-P | 1/100. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol. The mouse and rat recommendation is based on the WB results. This antibody may not be suitable for IHC with mouse or rat samples. |
|
ICC/IF | 1/250 - 1/500. |
ターゲット情報
-
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
-
参照データベース
- 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 -
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
-
All lanes : Anti-EGFR antibody [EP38Y] (ab52894) at 1/10000 dilution (purified)
Lane 1 : Rat liver lysates
Lane 2 : Mouse lung lysates
Lysates/proteins at 15 µg per lane.
Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution
Predicted band size: 134 kDa
Observed band size: 175 kDa why is the actual band size different from the predicted?Blocking and diluting buffer: 5% NFDM/TBST
-
All lanes : Anti-EGFR antibody [EP38Y] (ab52894) at 1/1000 dilution
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 - ab52894 observed at 175 kDa. Red - loading control, ab130007 observed at 125 kDa.
ab52894 was shown to react with EGFR in wild-type HeLa. 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. ab52894 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.
-
Anti-EGFR antibody [EP38Y] (ab52894) at 1/2000 dilution (purified) + HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates at 20 µg
Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution
Predicted band size: 134 kDa
Observed band size: 175 kDa why is the actual band size different from the predicted?Blocking and diluting buffer: 5% NFDM/TBST
-
Unpurified ab52894 stained A431 (Human epidermoid carcinoma cell line) cells.
The cells were fixed in 100% methanol for 5 mins at -20°C and then incubated in 1% BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1 hour at room temperature to permeabilize the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab52894 at 1 in 500) 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 1 hour at room temperature. Alexa Fluor® 594 WGA was used to label plasma membranes (pseudo-colored red) at a 1/200 dilution for 1 hour at room temperature. DAPI was used to stain the cell nuclei (pseudo-colored blue) at a concentration of 1.43 µM for 1 hour at room temperature.
-
All lanes : Anti-EGFR antibody [EP38Y] (ab52894) at 1/1000 dilution (unpurified)
Lane 1 : Caco-2 (Human colorectal adenocarcinoma cell line) cell lysate
Lane 2 : A431 (Human epidermoid carcinoma cell line) 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 mins before being transferred onto a Nitrocellulose membrane at 30V for 70 mins. The membrane was then blocked for an hour before being incubated with unpurified ab52894 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-labeled 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.
-
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-EGFR antibody [EP38Y] (ab52894)
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human cervical carcinoma tissue sections labeling EGFR with purified ab52894 at 1:100 dilution (0.95 μg/ml).
Heat mediated antigen retrieval was performed using EDTA buffer, pH 9.0. Tissue was counterstained with hematoxylin. ab97051 Goat Anti-Rabbit IgG H&L (HRP) secondary antibody was used at 1:500 dilution. PBS instead of the primary antibody was used as the negative control.
-
Immunocytochemistry/ Immunofluorescence analysis of A431 (Human epidermoid carcinoma epithelial cell) cells labeling EGFR with purified ab52894 at 1:250 dilution (0.4 μg/ml).
Cells were fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Cells were counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) 1:200 (2.5 μg/ml). ab150077 Goat anti rabbit IgG(Alexa Fluor® 488) was used as the secondary antibody at 1:1000 dilution. DAPI nuclear counterstain.
PBS instead of the primary antibody was used as the secondary antibody only control.
-
Flow Cytometry analysis of A431 (Human epidermoid carcinoma epithelial cell) cells labeling EGFR with purified ab52894.
Cells were fixed with 4% paraformaldehyde (10 mins) and permeabilized with 90% methanol for 30 mins. Then incubated in 1x PBS / 10% normal goat serum to block non-specific protein-protein interactions followed by ab52894 at 1/20 dilution (red) for 30 mins. A Goat anti rabbit IgG (Alexa Fluor® 488) secondary antibody was used at 1:2000 dilution. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).
-
ab52894 (purified) at 1:20 dilution (0.5 µg) immunoprecipitating EGFR in HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate.
Lane 1 (input): HeLa whole cell lysate 10 µg
Lane 2 (+): ab52894 in HeLa whole cell lysate
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab52894 in HeLa whole cell lysate
For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366) was used for detection at 1:1000 dilution.
Blocking and diluting buffer: 5% NFDM/TBST.
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.
参考文献 (174)
ab52894 は 174 報の論文で使用されています。
- Nakano Y et al. Clinical importance of long non-coding RNA LINC00460 expression in EGFR-mutant lung adenocarcinoma. Int J Oncol 56:243-257 (2020). PubMed: 31789388
- Strezoska Ž et al. CRISPR-mediated transcriptional activation with synthetic guide RNA. J Biotechnol 319:25-35 (2020). PubMed: 32470463
- Liu C et al. Lewis antigen-negative pancreatic cancer: An aggressive subgroup. Int J Oncol 56:900-908 (2020). PubMed: 32319567
- Song X et al. Zi Shen Huo Luo Formula Enhances the Therapeutic Effects of Angiotensin-Converting Enzyme Inhibitors on Hypertensive Left Ventricular Hypertrophy by Interfering With Aldosterone Breakthrough and Affecting Caveolin-1/Mineralocorticoid Receptor Colocalization and Downstream Extracellular Signal-Regulated Kinase Signaling. Front Pharmacol 11:383 (2020). PubMed: 32317965
- Deng QF et al. Cyclooxygenase-2 mediates gefitinib resistance in non-small cell lung cancer through the EGFR/PI3K/AKT axis. J Cancer 11:3667-3674 (2020). PubMed: 32284763