EGFR KO cell line available to order. KO validated by Next Generation Sequencing. Free of charge wild type control provided.
Images
Key facts
- Cell type
A549
- Species or organism
Human
- Tissue
Lung
- Form
Liquid
- Knockout validation
Next Generation Sequencing, Sanger Sequencing
Alternative names
Avian erythroblastic leukemia viral (v erb b) oncogene homolog, Cell growth inhibiting protein 40, Cell proliferation inducing protein 61, EGFR_HUMAN, ERBB, ERBB1, 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), Errp, HER1, NISBD2, Oncogen ERBB, PIG61, Proto-oncogene c-ErbB-1, Receptor tyrosine-protein kinase ErbB-1, SA7, Species antigen 7, Urogastrone, Wa5, erb-b2 receptor tyrosine kinase 1, mENA, v-erb-b Avian erythroblastic leukemia viral oncogen homolog, wa2
Recommended products
EGFR KO cell line available to order. KO validated by Next Generation Sequencing. Free of charge wild type control provided.
Key facts
- Cell type
A549
- Species or organism
Human
- Tissue
Lung
- Form
Liquid
- Knockout validation
Next Generation Sequencing, Sanger Sequencing
- Disease
Carcinoma
- Concentration
Properties
- Gene name
EGFR
- Gene editing type
Knockout
- Gene editing method
CRISPR technology
- Knockout validation
Next Generation Sequencing, Sanger Sequencing
Quality control
- STR analysis
CSF1PO, D13S317, D7S820, D5S818, TH01, D16S539, TPOX
Cell culture
- Biosafety level
EU: 1 US: 1
- Adherent/suspension
Adherent
- Gender
Male
Handling procedures
- Initial handling guidelines
Upon arrival, the vial should be stored in liquid nitrogen vapor phase and not at -80°C. Storage at -80°C may result in loss of viability.
1. Thaw the vial in 37°C water bath for approximately 1-2 minutes.
2. Transfer the cell suspension (0.8 mL) to a 15 mL/50 mL conical sterile polypropylene centrifuge tube containing 8.4 mL pre-warmed culture medium, wash vial with an additional 0.8 mL culture medium (total volume 10 mL) to collect remaining cells, and centrifuge at 201 x g (rcf) for 5 minutes at room temperature. 10 mL represents minimum recommended dilution. 20 mL represents maximum recommended dilution.
3. Resuspend the cell pellet in 5 mL pre-warmed culture medium and count using a haemocytometer or alternative cell counting method seed all remaining cells into a T25.
4. Incubate the culture at 37°C incubator with 5% CO2. Check the culture one day after revival and continue to check until 80% confluent. Media change can be given if needed.
5. Once confluent passage into an appropriate flask at a density of 2x104 cells/cm2. Seeding density is given as a guide only and should be scaled to align with individual lab schedules. Cultures should be monitored daily.- Subculture guidelines
- All seeding densities should be based on cell counts gained by established methods.
- A guide seeding density of 2x104 cells/cm2 is recommended.
- Cells should be passaged when they have achieved 80-90% confluence.
- Do not allow the cell density to exceed 7x104 cells/cm2.
- Culture medium
F-12K + 10% FBS
- Cryopreservation medium
Cell Freezing Medium-DMSO Serum free media, contains 8.7% DMSO in MEM supplemented with methyl cellulose.
Storage
- Shipped at conditions
Dry Ice
- Appropriate short-term storage conditions
-196°C
- Appropriate long-term storage conditions
-196°C
Notes
Recommended control: Human wild-type A549 cell line (Human wild-type A549 cell line ab288558). Please note a wild-type cell line is not automatically included with a knockout cell line order, if required please add recommended wild-type cell line at no additional cost using the code WILDTYPE-TMTK1.
Cryopreservation cell medium: Cell Freezing Medium-DMSO Serum free media, contains 8.7% DMSO in MEM supplemented with methyl cellulose.
Culture medium: F-12K + 10% FBS
Initial handling guidelines:
Upon arrival, the vial should be stored in liquid nitrogen vapor phase and not at -80°C. Storage at -80°C may result in loss of viability.
1. Thaw the vial in 37°C water bath for approximately 1-2 minutes.
2. Transfer the cell suspension (0.8 mL) to a 15 mL/50 mL conical sterile polypropylene centrifuge tube containing 8.4 mL pre-warmed culture medium, wash vial with an additional 0.8 mL culture medium (total volume 10 mL) to collect remaining cells, and centrifuge at 201 x g (rcf) for 5 minutes at room temperature. 10 mL represents minimum recommended dilution. 20 mL represents maximum recommended dilution.
3. Resuspend the cell pellet in 5 mL pre-warmed culture medium and count using a haemocytometer or alternative cell counting method. Based on cell count, seed cells in an appropriate cell culture flask at a density of 2x103-1x104 cells/cm2. Seeding density is given as a guide only and should be scaled to align with individual lab schedules.
4. Incubate the culture at 37°C incubator with 5% CO2. Cultures should be monitored daily.
Subculture guidelines:
- All seeding densities should be based on cell counts gained by established methods.
- A guide seeding density of 6x104 cells/cm2 is recommended.
- A partial media change 24 hours prior to subculture may be helpful to encourage growth, if required.
- Cells should be passaged when they have achieved 80-90% confluence.
- Do not allow the cell density to exceed 7x104 cells/cm2.
We will provide viable cells that proliferate on revival.
This product is subject to limited use licenses from The Broad Institute and ERS Genomics Limited, and is developed with patented technology. For full details of the limited use licenses and relevant patents please refer to our limited use license and patent pages.
Supplementary info
This supplementary information is collated from multiple sources and compiled automatically.
- Activity summary
EGFR or Epidermal Growth Factor Receptor is a transmembrane glycoprotein that acts as a receptor for members of the epidermal growth factor family. Known alternatively as ErbB1 or HER1 this receptor has an approximate molecular weight of 170 kDa. EGFR is expressed in various cell types notably on epithelial cells and can influence multiple cellular processes through its kinase activity. It participates in the regulation of cell growth multiplication and survival by activating its kinase domain upon ligand binding.
- Biological function summary
The EGFR protein plays an important role in cellular communication and signaling processes. EGFR pairs with other receptor family members to form active dimers or even higher-order complexes which in turn initiate intracellular signaling cascades. Through these complexes EGFR influences many processes including cell differentiation and repair. This function of EGFR makes it an integral part of mammalian biology affecting how cells respond to their environment by mediating changes in gene expression.
- Pathways
EGFR is a central player in the MAPK and PI3K/Akt signaling pathways. Alongside other protein partners like KRAS and PI3 kinase it contributes to transmitting signals from the cell surface to the nucleus affecting gene transcription and cell behavior. These pathways are important for normal cell growth and division and aberrations in these pathways can lead to excessive or insufficient cell proliferation.
- Associated diseases and disorders
EGFR is pertinent to cancer biology including non-small cell lung cancer and glioblastoma where mutations or overexpression of the receptor frequently occur. It connects to proteins such as PTEN and BRAF which influence tumor progression and response to targeted therapies. EGFR's involvement in these disorders highlights its significance as a therapeutic target since it can be manipulated to alter disease progression.
Product promise
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5 product images
Western blot - Human EGFR knockout A549 cell line (ab286394)
Western blot: Anti-EGFR antibody [E235] (Anti-EGFR antibody [E235] ab32077) staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, Anti-EGFR antibody [E235] ab32077 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 (Human EGFR knockout HeLa cell line 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.
All lanes: Western blot - Anti-EGFR antibody [E235] (Anti-EGFR antibody [E235] ab32077) at 1/1000 dilution
Lane 1: Wild-type A549 cell lysate at 20 µg
Lane 2: EGFR knockout A549 cell lysate at 20 µg
Lane 3: Wild-type HeLa cell lysate at 20 µg
Lane 4: EGFR knockout HeLa cell lysate at 20 µg
Performed under reducing conditions.
Observed band size: 160 kDa
Western blot - Human EGFR knockout A549 cell line (ab286394)
Western blot: Anti-EGFR antibody [E234] (Anti-EGFR antibody [E234] ab32198) staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, Anti-EGFR antibody [E234] 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 (Human EGFR knockout HeLa cell line 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.
All lanes: Western blot - Anti-EGFR antibody [E234] (Anti-EGFR antibody [E234] ab32198) at 1/1000 dilution
Lane 1: Wild-type A549 cell lysate at 20 µg
Lane 2: EGFR knockout A549 cell lysate at 20 µg
Lane 3: Wild-type HeLa cell lysate at 20 µg
Lane 4: EGFR knockout HeLa cell lysate at 20 µg
Performed under reducing conditions.
Observed band size: 160 kDa
Western blot - Human EGFR knockout A549 cell line (ab286394)
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (Anti-EGFR antibody [EP38Y] ab52894).
Western blot: Anti-EGFR antibody [EP38Y] (Anti-EGFR antibody [EP38Y] ab52894) staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, Anti-EGFR antibody [EP38Y] ab52894 was shown to bind specifically to EGFR. A band was observed at 175 kDa in wild-type A549 cell lysates with no signal observed at this size in EGFR knockout cell line. To generate this image, wild-type and EGFR knockout A549 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 5% 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.Lanes 1 - 4: Western blot - Anti-EGFR antibody [EP38Y] - BSA and Azide free (Anti-EGFR antibody [EP38Y] - BSA and Azide free ab272293)
Lanes 1 - 4: Western blot - Human EGFR knockout A549 cell line (ab286394)
Lane 1: Wild-type A549 cell lysate at 20 µg
Lane 2: EGFR knockout A549 cell lysate at 20 µg
Lane 3: Wild-type HeLa cell lysate at 20 µg
Lane 4: EGFR knockout HeLa cell lysate at 20 µg
Developed using the ECL technique.
Performed under reducing conditions.
Observed band size: 175 kDa
Next Generation Sequencing - Human EGFR knockout A549 cell line (ab286394)
6 bp (allele 1), 3 bp (allele 2), and 10 bp deletion after Ser160 of the WT protein
Sanger Sequencing - Human EGFR knockout A549 cell line (ab286394)
2 bp deletion after the Gln145 and 8 bp deletion after the Glu160 of the WT protein
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