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AB255394

Human HIF1A (HIF-1 alpha) knockout HCT116 cell line

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HIF1A KO cell line available to order. KO validated by Western blot. Free of charge wild type control available. Knockout achieved by using CRISPR/Cas9, Homozygous: 8 bp deletion in exon 3. To order both knockout and wild-type control cells: select 2 x 1000000Cells/vial. To order only knockout cells: select 1000000Cells/vial.

View Alternative Names

ARNT-interacting protein, Basic-helix-loop-helix-PAS protein MOP1, Class E basic helix-loop-helix protein 78, HIF-1, HIF-1-alpha, HIF-alpha, HIF1A_HUMAN, Hypoxia inducible factor 1 alpha isoform I.3, Hypoxia inducible factor 1 alpha subunit, Hypoxia inducible factor 1 alpha subunit basic helix loop helix transcription factor, Hypoxia inducible factor 1, alpha subunit (basic helix loop helix transcription factor), Hypoxia-inducible factor 1-alpha, MOP 1, Member of PAS protein 1, Member of PAS superfamily 1, Member of the PAS Superfamily 1, PAS domain-containing protein 8, PASD 8, bHLHe78, hifla

6 Images
Western blot - Human HIF1A (HIF-1 alpha) knockout HCT116 cell line (AB255394)
  • WB

Lab

Western blot - Human HIF1A (HIF-1 alpha) knockout HCT116 cell line (AB255394)

Western blot : Anti-HIF1A antibody [EP1215Y] (ab51608) staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab51608 was shown to bind specifically to HIF1A. A band was observed at 105 kDa in wild-type Wild-type HCT 116 DMOG (0 mM, 4 h) cell lysates with no signal observed at this size in HIF1A knockout cell line. To generate this image, wild-type and HIF1A knockout Wild-type HCT 116 DMOG (0 mM, 4 h) 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-HIF-1 alpha antibody [EP1215Y] (<a href='/en-us/products/primary-antibodies/hif-1-alpha-antibody-ep1215y-ab51608'>ab51608</a>) at 1/1000 dilution

Lane 1:

Wild-type HCT116 DMOG (0 mM, 4 h) cell lysate at 20 µg

Lane 2:

Wild-type HCT116 treated DMOG (1 mM, 4 h) cell lysate at 20 µg

Lane 3:

HIF1A knockout HCT116 DMOG (0 mM, 4 h) cell lysate at 20 µg

Lane 4:

HIF1A knockout HCT116 treated DMOG (1 mM, 4 h) cell lysate at 20 µg

Secondary

All lanes:

Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Observed band size: 105 kDa

false

Western blot - Human HIF1A (HIF-1 alpha) knockout HCT116 cell line (AB255394)
  • WB

Lab

Western blot - Human HIF1A (HIF-1 alpha) knockout HCT116 cell line (AB255394)

False colour image of Western blot : Anti-HIF-1 alpha antibody [54/HIF-1a] staining at 1/2000 dilution shown in green; Rabbit anti-alpha Tubulin antibody [EP1332Y] (ab52866) loading control staining at 1/20000 dilution shown in red. In Western blot ab279654 was shown to bind specifically to HIF-1 alpha. A band was observed at 110 kDa in treated wild-type HCT 116 cell lysates with no signal observed at this size in HIF1A knockout cell line ab255394 (knockout cell lysate ab263860). To generate this image wild-type and HIF1A knockout HCT 116 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-Mouse IgG H&L (IRDye® 800CW) preabsorbed (ab216772) and Goat anti-Rabbit IgG H&L (IRDye® 680RD) preabsorbed (ab216777) at 1/20000 dilution.

All lanes:

Western blot - Anti-HIF-1 alpha antibody [54/HIF-1a] (<a href='/en-us/products/primary-antibodies/hif-1-alpha-antibody-54-hif-1a-ab279654'>ab279654</a>) at 1/2000 dilution

Lane 1:

Wild-type HCT 116 Untreated DMOG Control cell lysate at 20 µg

Lane 2:

Wild-type HCT 116 Treated DMOG (1 mM, 4 h) cell lysate at 20 µg

Lane 3:

HIF1A knockout HCT 116 Untreated DMOG Control cell lysate at 20 µg

Lanes 3 - 4:

Western blot - Human HIF1A (HIF-1 alpha) knockout HCT116 cell line (ab255394)

Lane 4:

HIF1A knockout HCT 116 Treated DMOG (1 mM, 4 h) cell lysate at 20 µg

Predicted band size: 92 kDa

false

Western blot - Human HIF1A (HIF-1 alpha) knockout HCT116 cell line (AB255394)
  • WB

Lab

Western blot - Human HIF1A (HIF-1 alpha) knockout HCT116 cell line (AB255394)

Western blot : Anti-HIF1A antibody [EP1215Y] (ab51608) staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab51608 was shown to bind specifically to HIF1A. A band was observed at 100 kDa in wild-type HCT 116 cell lysates with no signal observed at this size in HIF1A knockout cell line. To generate this image, wild-type and HIF1A knockout HCT 116 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-HIF-1 alpha antibody [EP1215Y] (<a href='/en-us/products/primary-antibodies/hif-1-alpha-antibody-ep1215y-ab51608'>ab51608</a>) at 1/1000 dilution

Lane 1:

Wild-type HCT 116 DMOG (0 mM, 4 h) nuclear cell lysate at 20 µg

Lane 2:

Wild-type HCT 116 treated DMOG (1 mM, 4 h) nuclear cell lysate at 20 µg

Lane 3:

HIF1A knockout HCT 116 DMOG (0 mM, 4 h) nuclear cell lysate at 20 µg

Lane 4:

HIF1A knockout HCT 116 treated DMOG (1 mM, 4 h) nuclear cell lysate at 20 µg

Lane 5:

Wild-type HCT 116 DMOG (0 mM, 4 h) cell lysate at 20 µg

Lane 6:

Wild-type HCT 116 treated DMOG (1 mM, 4 h) cell lysate at 20 µg

Lane 7:

HIF1A knockout HCT 116 DMOG (0 mM, 4 h) cell lysate at 20 µg

Lane 8:

HIF1A knockout HCT 116 treated DMOG (1 mM, 4 h) cell lysate at 20 µg

Secondary

All lanes:

Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Observed band size: 100 kDa

false

Western blot - Human HIF1A (HIF-1 alpha) knockout HCT116 cell line (AB255394)
  • WB

Lab

Western blot - Human HIF1A (HIF-1 alpha) knockout HCT116 cell line (AB255394)

Western blot : Anti-HIF1A antibody [H1alpha67] (ab1) staining at 5 ug/ml, shown in green; Rabbit anti-alpha Tubulin antibody [EP1332Y] (ab52866) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab1 was shown to bind specifically to HIF1A. A band was observed at 100 kDa in wild-type HCT 116 cell lysates with no signal observed at this size in HIF1A knockout cell line. To generate this image, wild-type and HIF1A knockout HCT 116 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-Mouse IgG H&L 800CW and Goat anti-Rabbit IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-HIF-1 alpha antibody [H1alpha67] (<a href='/en-us/products/primary-antibodies/hif-1-alpha-antibody-h1alpha67-ab1'>ab1</a>) at 5 µg/mL

Lane 1:

Wild-type HCT 116 DMOG (0 mM, 4 h) nuclear cell lysate at 20 µg

Lane 2:

Wild-type HCT 116 treated DMOG (1 mM, 4 h) nuclear cell lysate at 20 µg

Lane 3:

HIF1A knockout HCT 116 DMOG (0 mM, 4 h) nuclear cell lysate at 20 µg

Lane 4:

HIF1A knockout HCT 116 treated DMOG (1 mM, 4 h) nuclear cell lysate at 20 µg

Lane 5:

Wild-type HCT 116 DMOG (0 mM, 4 h) cell lysate at 20 µg

Lane 6:

Wild-type HCT 116 treated DMOG (1 mM, 4 h) cell lysate at 20 µg

Lane 7:

HIF1A knockout HCT 116 DMOG (0 mM, 4 h) cell lysate at 20 µg

Lane 8:

HIF1A knockout HCT 116 treated DMOG (1 mM, 4 h) cell lysate at 20 µg

Secondary

All lanes:

Goat anti-Mouse IgG H&L 800CW and Goat anti-Rabbit IgG H&L 680RD at 1/20000 dilution

Observed band size: 100 kDa

false

Sanger Sequencing - Human HIF1A (HIF-1 alpha) knockout HCT116 cell line (AB255394)
  • Sanger seq

Unknown

Sanger Sequencing - Human HIF1A (HIF-1 alpha) knockout HCT116 cell line (AB255394)

Homozygous : 8 bp deletion in exon3

Immunocytochemistry/ Immunofluorescence - Human HIF1A (HIF-1 alpha) knockout HCT116 cell line (AB255394)
  • ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Human HIF1A (HIF-1 alpha) knockout HCT116 cell line (AB255394)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HIF1A KO HCT116(HIF1A knockout human colorectal carcinoma epithelial cell),ab255394 cells labelling HIF-1 alpha with ab317044 at 1/200 (2.53 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2 ug/ml dilution (Green).

Confocal image showing negative staining in both HIF1A knockout HCT 116 cells and wildtype HCT 116 cells, the nuclear signal was increased in wildtype HCT 116 cells treated with 0.5 mM DFO for 24 hours (shown in green). The counterstain was observed in red. Nuclear DNA was labelled with DAPI (shown in blue).
Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8).

ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5ug/ml dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/ml dilution.

Key facts

Cell type

HCT116

Species or organism

Human

Tissue

Colon

Form

Liquid

form

Knockout validation

Sanger Sequencing,Western blot

Mutation description

Knockout achieved by using CRISPR/Cas9, Homozygous: 8 bp deletion in exon 3

Disease

Carcinoma

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Reactivity data

{ "title": "Reactivity Data", "filters": { "stats": ["", "Reactivity", "Dilution Info", "Notes"] }, "values": { "WB": { "reactivity":"TESTED_AND_REACTS", "dilution-info":"", "notes":"<p></p>" } } }
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Product details

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.

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What's included?

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Properties and storage information

Gene name
HIF1A
Gene editing type
Knockout
Gene editing method
CRISPR technology
Knockout validation
Sanger Sequencing, Western blot
Zygosity
Homozygous
Shipped at conditions
Dry Ice
Appropriate short-term storage conditions
-196°C
Appropriate long-term storage conditions
-196°C
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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.
Culture medium

McCoY5a + 10% FBS

Cryopreservation medium

Cell Freezing Medium-DMSO Serum free media, contains 8.7% DMSO in MEM supplemented with methyl cellulose.

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Supplementary information

This supplementary information is collated from multiple sources and compiled automatically.

HIF-1 alpha also known as hypoxia-inducible factor 1-alpha is a transcription factor critical in cellular response to low oxygen levels. Its molecular weight usually ranges from 93 to 120 kDa. You can find HIF-1 alpha expressed in tissues throughout the body but its expression significantly increases under hypoxic conditions. Researchers often use the HIF-1a ELISA to measure its expression levels. HIF-1 alpha forms a complex with other proteins to perform its functions effectively.
Biological function summary

HIF-1 alpha regulates gene expression in response to hypoxic conditions in cells. It forms a complex with HIF-1 beta to activate transcription of various genes involved in energy metabolism angiogenesis and erythropoiesis. HIF-1 alpha enables cells to adapt to reduced oxygen availability allowing for cellular survival and function under stress. It plays an important role in promoting the expression of genes like VEGF and EPO which are important for vascular and red blood cell development respectively.

Pathways

HIF-1 alpha plays an integral role in the hypoxia signaling pathway and the glycolytic pathway. In the hypoxia signaling pathway HIF-1 alpha partners with VHL (Von Hippel-Lindau) protein that regulates its degradation under normal oxygen conditions. When oxygen levels drop HIF-1 alpha avoids degradation stabilizes and translocates into the nucleus to initiate transcription of hypoxia-responsive genes. The glycolytic pathway involvement highlights its function in adapting energy production under hypoxic conditions through collaboration with enzymes and transporters associated with glycolysis.

HIF-1 alpha has been implicated in cancer and ischemic diseases. Its role in promoting angiogenesis and metabolic adaptation makes it a contributor to tumor growth and survival collaborating with oncogenes such as c-Myc. In ischemic diseases like stroke or myocardial infarction HIF-1 alpha's ability to induce protective responses can mitigate tissue damage through regulation of survival pathways. Understanding these interactions helps in the development of therapeutic strategies targeting HIF-1 alpha in disease contexts.
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Quality control

STR analysis

CSF1PO, D13S317, D7S820, D5S818, TH01, D16S539, TPOX

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Cell culture

Biosafety level

EU: 1 US: 1

Adherent/suspension

Adherent

Gender

Male

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Product protocols

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Target data

See full target information HIF1A
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