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    products/cell-lysates/human-egfr-knockout-hela-cell-lysate-ab263845.pdf

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Signal Transduction Protein Phosphorylation Tyrosine Kinases Receptor Tyrosine Kinases
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Human EGFR knockout HeLa cell lysate (ab263845)

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Western blot - Human EGFR knockout HeLa cell lysate (ab263845)
  • Western blot - Human EGFR knockout HeLa cell lysate (ab263845)
  • Sanger Sequencing - Human EGFR knockout HeLa cell lysate (ab263845)
  • Sanger Sequencing - Human EGFR knockout HeLa cell lysate (ab263845)

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Overview

  • Product name

    Human EGFR knockout HeLa cell lysate
    See all EGFR kits
  • Product overview


    Knockout cell lysate achieved by CRISPR/Cas9.

  • Parental Cell Line

    HeLa
  • Organism

    Human
  • Mutation description

    Knockout achieved by using CRISPR/Cas9, 1 bp deletion in exon1 and 1 bp insertion in exon1.
  • Passage number

    <20
  • Knockout validation

    Sanger Sequencing, Western Blot (WB)
  • Reconstitution notes

    To use as WB control, resuspend the lyophilizate in 50 µL of LDS* Sample Buffer to have a final concentration of 2 mg/ml. For reducing conditions, we recommend a final concentration of 0.1 M DTT.

    *Usage of SDS sample buffer is not recommended with these lyophilized lysates.

  • Notes

    Lysate preparation: Our lysates are made using RIPA buffer to which we add a protease inhibitor cocktail and phosphatase inhibitor cocktail (ratio: 300:100:10). This means that the protein of interest is denatured. If you require a native form of the protein please use the live cell version - found here. Please refer to our lysis protocol for further details on how our lysates are prepared.

    User storage instructions: Lyophilizate may be stored at 4°C. After reconstitution, store at -20°C for short-term storage or -80°C for long-term storage.

    Access thousands of knockout cell lysates, generated from commonly used cancer cell lines.
    See here for more information on knockout cell lysates.

    Abcam has not and does not intend to apply for the REACH Authorisation of customers’ uses of products that contain European Authorisation list (Annex XIV) substances.
    It is the responsibility of our customers to check the necessity of application of REACH Authorisation, and any other relevant authorisations, for their intended uses.

    This product is subject to limited use licenses from The Broad Institute, ERS Genomics Limited and Sigma-Aldrich Co. LLC, and is developed with patented technology. For full details of the licenses and patents please refer to our limited use license and patent pages.

  • Tested applications

    Suitable for: WBmore details

Properties

  • Storage instructions

    Store at -80°C. Please refer to protocols.
  • Components 1 kit
    ab255489 - Human EGFR knockout HeLa cell lysate 1 x 100µg
    ab255929 - Human wild-type HeLa cell lysate 1 x 100µg
  • Research areas

    • Signal Transduction
    • Protein Phosphorylation
    • Tyrosine Kinases
    • Receptor Tyrosine Kinases
    • Signal Transduction
    • Growth Factors/Hormones
    • EGF
    • Cell Biology
    • Cell Cycle
    • Cell differentiation
    • Microbiology
    • Organism
    • Virus
    • RNA Virus
    • ssRNA positive strand virus
    • SARS Coronavirus
    • Cancer
    • Growth factors
    • EGF
    • Cancer
    • Signal transduction
    • Protein phosphorylation
    • Tyrosine kinases
    • Receptor tyrosine kinases
    • Cancer
    • Oncoproteins/suppressors
    • Oncoproteins
    • Growth factor receptors
    • Cancer
    • Tumor biomarkers
    • Receptors
    • Neuroscience
    • Development
    • Neuroscience
    • Processes
  • Cell type

    epithelial
  • Disease

    Adenocarcinoma
  • Gender

    Female
  • STR Analysis

    Amelogenin X D5S818: 11, 12 D13S317: 12, 13.3 D7S820: 8, 12 D16S539: 9, 10 vWA: 16, 18 TH01: 7 TPOX: 8,12 CSF1PO: 9, 10

Target

  • Function

    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.
  • Tissue specificity

    Ubiquitously expressed. Isoform 2 is also expressed in ovarian cancers.
  • Involvement in disease

    Lung cancer
    Inflammatory skin and bowel disease, neonatal, 2
  • Sequence similarities

    Belongs to the protein kinase superfamily. Tyr protein kinase family. EGF receptor subfamily.
    Contains 1 protein kinase domain.
  • Post-translational
    modifications

    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.
  • Cellular localization

    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).
  • Target information above from: UniProt accession P00533 The UniProt Consortium
    The Universal Protein Resource (UniProt) in 2010
    Nucleic Acids Res. 38:D142-D148 (2010) .

    Information by UniProt
  • Alternative names

    • Avian erythroblastic leukemia viral (v erb b) oncogene homolog
    • Cell growth inhibiting protein 40
    • Cell proliferation inducing protein 61
    • EGF R
    • EGFR
    • EGFR_HUMAN
    • Epidermal growth factor receptor
    • Epidermal growth factor receptor (avian erythroblastic leukemia viral (v erb b) oncogene homolog)
    • Epidermal growth factor receptor (erythroblastic leukemia viral (v erb b) oncogene homolog avian)
    • erb-b2 receptor tyrosine kinase 1
    • ERBB
    • ERBB1
    • Errp
    • HER1
    • mENA
    • NISBD2
    • Oncogen ERBB
    • PIG61
    • Proto-oncogene c-ErbB-1
    • Receptor tyrosine protein kinase ErbB 1
    • Receptor tyrosine-protein kinase ErbB-1
    • SA7
    • Species antigen 7
    • Urogastrone
    • v-erb-b Avian erythroblastic leukemia viral oncogen homolog
    • wa2
    • Wa5
    see all

Associated products

  • KO cell lines

    • Human EGFR knockout HeLa cell line (ab255385)
  • Related Products

    • Anti-EGFR antibody [EP38Y] - Low endotoxin, Azide free (ab174481)
    • Anti-EGFR antibody [E235] - BSA and Azide free (ab227459)
    • Anti-EGFR antibody [E235] (ab32077)
    • Anti-EGFR antibody [EP38Y] (ab52894)

Applications

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab263845 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
WB
Use at an assay dependent concentration.
Notes
WB
Use at an assay dependent concentration.

Images

  • Western blot - Human EGFR knockout HeLa cell lysate (ab263845)
    Western blot - Human EGFR knockout HeLa cell lysate (ab263845)

    Lane 1: A431 cell lysate (20 µg)

    Lane 2: MDA-MB-468 cell lysate (20 µg)

    Lane 3: Wild-type HeLa cell lysate (20 µg)

    Lane 4: EGFR knockout HeLa cell lysate (20 µg)

    Lanes 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.

  • Western blot - Human EGFR knockout HeLa cell lysate (ab263845)
    Western blot - Human EGFR knockout HeLa cell lysate (ab263845)

    Lane 1: A431 cell lysate (20 µg)

    Lane 2: MDA-MB-468 cell lysate (20 µg)

    Lane 3: Wild-type HeLa cell lysate (20 µg)

    Lane 4: EGFR knockout HeLa cell lysate (20 µg)

    Lanes 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.

  • Sanger Sequencing - Human EGFR knockout HeLa cell lysate (ab263845)
    Sanger Sequencing - Human EGFR knockout HeLa cell lysate (ab263845)

    Allele-1: 1 bp deletion in exon1

     

  • Sanger Sequencing - Human EGFR knockout HeLa cell lysate (ab263845)
    Sanger Sequencing - Human EGFR knockout HeLa cell lysate (ab263845)

    Allele-2: 1 bp insertion in exon1

     

Protocols

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.

Click here to view the general protocols

Datasheets and documents

  • SDS download

  • Datasheet download

    Download

References (0)

Publishing research using ab263845? Please let us know so that we can cite the reference in this datasheet.

ab263845 has not yet been referenced specifically in any publications.

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