Human IRF9 (Interferon regulatory factor 9) knockout HeLa cell line (ab266051)
Overview
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Product name
Human IRF9 (Interferon regulatory factor 9) knockout HeLa cell line
See all Interferon regulatory factor 9/IRF-9 lysates -
Parental Cell Line
HeLa -
Organism
Human -
Mutation description
Knockout achieved by using CRISPR/Cas9, 1 bp insertion in exon 2 and 2 bp deletion in exon 2 -
Passage number
<20 -
Knockout validation
Immunocytochemistry (ICC), Sanger Sequencing -
Tested applications
Suitable for: ICC, WBmore details -
Biosafety level
2 -
General notes
Recommended control: Human wild-type HeLa cell line (ab255928). 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: DMEM (High Glucose) + 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 2x104 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 2x104 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.
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.
We will provide viable cells that proliferate on revival.
Properties
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Number of cells
1 x 106 cells/vial, 1 mL -
Adherent /Suspension
Adherent -
Tissue
Cervix -
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 -
Mycoplasma free
Yes -
Storage instructions
Shipped on Dry Ice. Store in liquid nitrogen. -
Storage buffer
Constituents: 8.7% Dimethylsulfoxide, 2% Cellulose, methyl ether -
Research areas
Target
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Function
Transcription regulatory factor that mediates signaling by type I IFNs (IFN-alpha and IFN-beta). Following type I IFN binding to cell surface receptors, Jak kinases (TYK2 and JAK1) are activated, leading to tyrosine phosphorylation of STAT1 and STAT2. The phosphorylated STATs dimerize, associate with IRF9/ISGF3G to form a complex termed ISGF3 transcription factor, that enters the nucleus. ISGF3 binds to the IFN stimulated response element (ISRE) to activate the transcription of interferon stimulated genes, which drive the cell in an antiviral state. -
Sequence similarities
Belongs to the IRF family.
Contains 1 IRF tryptophan pentad repeat DNA-binding domain. -
Cellular localization
Cytoplasm. Nucleus. Translocated into the nucleus upon activation by IFN-alpha/beta. - Information by UniProt
Associated products
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KO cell lysates
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab266051 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 |
|---|---|---|
| ICC |
Use at an assay dependent concentration.
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| WB |
Use at an assay dependent concentration. Predicted molecular weight: 44 kDa.
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| Notes |
|---|
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ICC
Use at an assay dependent concentration. |
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WB
Use at an assay dependent concentration. Predicted molecular weight: 44 kDa. |
Images
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Western blot - Human IRF9 (Interferon regulatory factor 9) knockout HeLa cell line (ab266051)All lanes : Anti-Interferon regulatory factor 9/IRF-9 antibody [EPR24260-55] (ab271043) at 1/1000 dilution
Lane 1 : wild-type HeLa Treated Interferon-alpha1 (hIFN-a1) (10 ng/ml, 16 h) cell lysate
Lane 2 : IRF9 knockout HeLa treated: hIFN-a1 (10 ng/ml, 16 h) cell lysate
Lane 3 : wild-type HeLa Control Interferon-alpha1 (hIFN-a1) (0 ng/ml, 16 h) cell lysate
Lane 4 : IRF9 knockout HeLa vehicle control: hIFN-a1 (0 ng/ml, 16 h) cell lysate
Lane 5 : THP-1 cell lysate
Lane 6 : ACHN cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 44 kDa
Observed band size: 48 kDa why is the actual band size different from the predicted?False colour image of Western blot: Anti-Interferon regulatory factor 9/IRF-9 antibody [EPR24260-55] staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab271043 was shown to bind specifically to Interferon regulatory factor 9/IRF-9. A band was observed at 48 kDa in treated wild-type HeLa cell lysates with no signal observed at this size in IRF9 knockout cell line ab266051 (knockout cell lysate ab258472). The band observed in the knockout lysate lane below 48 kDa is likely to represent a truncated form of Interferon regulatory factor 9/IRF-9. This has not been investigated further and the functional properties of the gene product have not been determined. To generate this image, wild-type and IRF9 knockout HeLa cell lysates were analysed.First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution 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 (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) at 1/20000 dilution.
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Immunocytochemistry - Human IRF9 (Interferon regulatory factor 9) knockout HeLa cell line (ab266051)ab271043 staining Interferon regulatory factor 9 in wild-type HeLa cells and IRF9 knockout HeLa cells treated with interferon-a1 (ab48750) at 10 ng/ml for 16 hours. The cells were fixed with 4% paraformaldehyde (10 min) then permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab271043 at 0.4 μg/ml concentration and ab7291 (Mouse monoclonal to alpha Tubulin) at 1/1000 dilution overnight at 4°C followed by a further incubation at room temperature for 1h with a goat secondary antibody to rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to mouse IgG (Alexa Fluor® 594) (ab150120) at 2 μg/ml (shown in red). Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems TCS SP8). -
Western blot - Human IRF9 (Interferon regulatory factor 9) knockout HeLa cell line (ab266051)All lanes : Anti-Interferon regulatory factor 9/IRF-9 antibody [14HCLC] (ab277803) at 1/1000 dilution
Lane 1 : Wild-type HeLa treated hIFN-a1 (10 ng/ml, 16 h) cell lysate
Lane 2 : IRF9 knockout HeLa treated hIFN-a1 (10 ng/ml, 16 h) cell lysate
Lane 3 : Wild-type HeLa vehicle control hIFN-a1 (0 ng/ml, 16 h) cell lysate
Lane 4 : IRF9 knockout HeLa vehicle control hIFN-a1 (0 ng/ml, 16 h) cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 44 kDa
Observed band size: 48 kDa why is the actual band size different from the predicted?False colour image of Western blot: Anti-Interferon regulatory factor 9/IRF-9 antibody [14HCLC] staining at 2 µg/ml, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab277803 was shown to bind specifically to Interferon regulatory factor 9/IRF-9. A band was observed at 48 kDa in treated wild-type HeLa cell lysates with no signal observed at this size in IRF9 knockout cell line ab266051 (knockout cell lysate ab258472). The band observed in the knockout lysate lane below 48 kDa is likely to represent a truncated form of Interferon regulatory factor 9/IRF-9. This has not been investigated further and the functional properties of the gene product have not been determined. To generate this image, wild-type and IRF9 knockout HeLa 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 (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) at 1/20000 dilution.
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Sanger Sequencing - Human IRF9 knockout HeLa cell line (ab266051)Allele-1: 1 bp insertion in exon2
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Western blot - Human IRF9 (Interferon regulatory factor 9) knockout HeLa cell line (ab266051)All lanes : Anti-IRF9 antibody at 1/1000 dilution
Lane 1 : Wild-type HeLa cell lysate
Lane 2 : IRF9 knockout HeLa cell lysate
Lane 3 : THP-1 cell lysate
Lane 4 : ACHN cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 44 kDa
Observed band size: 48 kDa why is the actual band size different from the predicted?Lanes 1 - 4: Merged signal (red and green). Green - Anti-IRF9 antibody observed at 48 kDa. Red - loading control, ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37 kDa.
Anti-IRF9 antibody was shown to react with IRF-9 in wild-type HeLa cells in western blot. The bands observed in IRF9 knockout cell line ab266051 (IRF9 knockout cell lysate ab258472) below 48 kDa may represent truncated forms and cleaved fragments. This has not been investigated further and the functional properties of the gene product have not been determined. HeLa wild-type and IRF9 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween®) before incubation with anti-IRF9 antibody and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4°C at a 1 in 1000 dilution 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 h at room temperature before imaging.
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Sanger Sequencing - Human IRF9 knockout HeLa cell line (ab266051)Allele-2: 2 bp deletion in exon 2.
Protocols
Datasheets and documents
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SDS download
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Datasheet download
References (0)
ab266051 has not yet been referenced specifically in any publications.