Human EPAS1 (HIF-2-alpha) knockout A549 cell line
- Advanced Validation
- What is this?
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EPAS1 KO cell line available to order. KO validated by Next Generation Sequencing, Western blot. Free of charge wild type control provided. Knockout achieved by CRISPR/Cas9 X = 1 bp insertion Frameshift = 99%.
View Alternative Names
Basic-helix-loop-helix-PAS protein MOP2, Class E basic helix-loop-helix protein 73, ECYT4, EPAS1_HUMAN, Endothelial PAS domain-containing protein 1, Endothelial pas domain protein 1, HIF-1-alpha-like factor, HIF-2-alpha, HIF2A, Hypoxia inducible factor 2 alpha subunit, Hypoxia-inducible factor 2-alpha, MOP 2, Member of PAS protein 2, Member of pas superfamily 2, PAS domain-containing protein 2, PASD2, bHLHe73
- WB
Lab
Western blot - Human EPAS1 (HIF-2-alpha) knockout A549 cell line (AB259774)
Lanes 1 - 4 : Merged signal (red and green). Green - ab109616 observed at 100 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.
ab109616 was shown to react with HIF-2-alpha in A549 wild-type cells in western blot with loss of signal observed in EPAS1 knockout sample. A549 wild-type and EPAS1 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween®) before incubation with ab109616 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4° at 1 μg/ml and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.
All lanes:
Western blot - Anti-HIF-2-alpha antibody (<a href='/en-us/products/primary-antibodies/hif-2-alpha-antibody-ab109616'>ab109616</a>) at 1 µg/mL
Lane 1:
Wild-type A549 untreated cell lysate at 40 µg
Lane 2:
Wild-type A549 + DFO (1mM, 24 hours) cell lysate at 40 µg
Lane 2:
Western blot - Human EPAS1 (HIF-2-alpha) knockout A549 cell line (ab259774)
Lane 3:
EPAS knockout A549 untreated cell lysate at 40 µg
Lane 4:
EPAS knockout A549 + DFO (1mM, 24 hours) cell lysate at 40 µg
Predicted band size: 96 kDa
Observed band size: 100 kDa
false
- WB
Supplier Data
Western blot - Human EPAS1 (HIF-2-alpha) knockout A549 cell line (AB259774)
False colour image of Western blot : Anti-HIF-2-alpha antibody [OTI2G5] staining at 1/500 dilution shown in black; Rabbit Anti-GAPDH antibody [EPR16891] (ab181602) loading control staining at 1/20000 dilution shown in red. In Western blot ab157249 was shown to bind specifically to HIF-2-alpha. A band was observed at 100 kDa in treated wild-type A549 cell lysates with no signal observed at this size in EPAS1 knockout cell line ab259774 (knockout cell lysate ab259779). To generate this image wild-type and EPAS1 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 % BSA in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °. Blots were washed four times in TBS-T incubated with secondary antibodies for 1 h at room temperature washed again four times before development with Optiblot (ECL reagent ab133456) and imaged with 20 seconds exposure time. Secondary antibodies used were HRP conjugated Goat anti-Mouse (H+L) and Goat anti-Rabbit IgG H&L (IRDye® 680RD) preabsorbed (ab216777) at 1/20000 dilution.
All lanes:
Western blot - Anti-HIF-2-alpha antibody [OTI2G5] (<a href='/en-us/products/primary-antibodies/hif-2-alpha-antibody-oti2g5-ab157249'>ab157249</a>) at 1/500 dilution
Lane 1:
Wild-type A549 Untreated (DFO Control) cell lysate at 20 µg
Lane 2:
Wild-type A549 Treated DFO (1 mM, 24 h) cell lysate at 20 µg
Lane 2:
Western blot - Human EPAS1 (HIF-2-alpha) knockout A549 cell line (ab259774)
Lane 3:
EPAS1 knockout A549 Untreated (DFO Control) cell lysate at 20 µg
Lane 4:
EPAS1 knockout A549 Treated DFO (1 mM, 24 h) cell lysate at 20 µg
Predicted band size: 96 kDa
Observed band size: 100 kDa
false
- NGS
Lab
Next Generation Sequencing - Human EPAS1 (HIF-2-alpha) knockout A549 cell line (AB259774)
X = 1 bp insertion
Reactivity data
Product details
Recommended control: Human wild-type A549 cell line (ab255450). 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.
We will provide viable cells that proliferate on revival.
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.
What's included?
Properties and storage information
Gene name
Gene editing type
Gene editing method
Knockout validation
Shipped at conditions
Appropriate short-term storage conditions
Appropriate long-term storage conditions
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.
Supplementary information
This supplementary information is collated from multiple sources and compiled automatically.
Biological function summary
HIF-2-alpha regulates the expression of genes involved in energy metabolism and angiogenesis. The protein forms a complex by dimerizing with the HIF-1-beta subunit which is necessary for transcriptional activity. Through this complex formation and activity it influences processes such as erythropoiesis and regulates factors like vascular endothelial growth factor (VEGF). Therefore HIF-2-alpha contributes to the adaptation of cells and tissues under hypoxic conditions.
Pathways
HIF-2-alpha engages in the hypoxia signaling pathway playing an essential part by modulating gene expression in response to low oxygen availability. This role impacts other proteins such as HIF-1-alpha sharing overlapping functions but with distinct target genes. Additionally HIF-2-alpha is involved in the mTOR pathway which influences cell growth and metabolism through its interaction with the nutrient-sensing regulatory pathway.
Quality control
STR analysis
CSF1PO, D13S317, D7S820, D5S818, TH01, D16S539, TPOX
Cell culture
Biosafety level
EU: 1 US: 1
Adherent/suspension
Adherent
Gender
Male
Target data
Complementary Products
Cell Lines & Lysates
AB255394
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Advanced Validation
- 5
- 2
![Western blot - Anti-HIF-2-alpha antibody [EPR19656] (AB207607)](https://content.abcam.com/products/images/hif-2-alpha-antibody-epr19656-ab207607--western-blot-img21308.jpg)
Product promise
Please note: All products are 'FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES'.
For licensing inquiries, please contact partnerships@abcam.com