Human IL6ST (CD130/gp130) knockout A549 cell line (ab266938)
Overview
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Product name
Human IL6ST (CD130/gp130) knockout A549 cell line
See all CD130 (gp130) lysates -
Parental Cell Line
A549 -
Organism
Human -
Mutation description
Knockout achieved by using CRISPR/Cas9, 1 bp insertion in exon 4 and 4 bp deletion in exon 4 -
Passage number
<20 -
Knockout validation
Sanger Sequencing, Western Blot (WB) -
Tested applications
Suitable for: WBmore details -
Biosafety level
2 -
General notes
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.
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 exceed 7x104 cells/cm2.
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
Lung -
Cell type
epithelial -
Disease
Carcinoma -
Gender
Male -
STR Analysis
Amelogenin X,Y D5S818: 11 D13S317: 11 D7S820: 8, 11 D16S539: 11, 12 vWA: 14 TH01: 8,9.3 TPOX: 8,11 CSF1PO: 10, 12 -
Antibiotic resistance
Puromycin 1.00µg/ml -
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
Signal-transducing molecule. The receptor systems for IL6, LIF, OSM, CNTF, IL11, CTF1 and BSF3 can utilize gp130 for initiating signal transmission. Binds to IL6/IL6R (alpha chain) complex, resulting in the formation of high-affinity IL6 binding sites, and transduces the signal. Does not bind IL6. May have a role in embryonic development (By similarity). The type I OSM receptor is capable of transducing OSM-specific signaling events. -
Tissue specificity
Found in all the tissues and cell lines examined. Expression not restricted to IL6 responsive cells. -
Sequence similarities
Belongs to the type I cytokine receptor family. Type 2 subfamily.
Contains 5 fibronectin type-III domains.
Contains 1 Ig-like C2-type (immunoglobulin-like) domain. -
Domain
The WSXWS motif appears to be necessary for proper protein folding and thereby efficient intracellular transport and cell-surface receptor binding.
The box 1 motif is required for JAK interaction and/or activation. -
Post-translational
modificationsPhosphorylation of Ser-782 down-regulates cell surface expression.
Heavily N-glycosylated. -
Cellular localization
Secreted and Cell membrane. - Information by UniProt
Associated products
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KO cell lysates
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Related Products
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab266938 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 |
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WB |
Use at an assay dependent concentration. Predicted molecular weight: 104 kDa.
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Notes |
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WB
Use at an assay dependent concentration. Predicted molecular weight: 104 kDa. |
Images
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All lanes : Anti-CD130 (gp130) antibody [EPR24557-50] (ab283685) at 1/1000 dilution
Lane 1 : Wild-type A549 (human lung carcinoma epithelial cell) whole cell lysate
Lane 2 : IL6ST (CD130/gp130) knockout A549 (human lung carcinoma epithelial cell) whole cell lysate
Lane 3 : PC-3 (human prostate adenocarcinoma epithelial cell) whole cell lysate
Lysates/proteins at 20 µg per lane.
Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)
Predicted band size: 104 kDa
Observed band size: 130 kDa why is the actual band size different from the predicted?Blocking and diluting buffer and concentration: 5% NFDM/TBST
ab283685 was shown to specifically react with IL6ST (CD130/gp130) in wild-type A549 cells. Loss of signal was observed when knockout cell line ab266938 (knockout cell lysate ab257207) was used. Wild-type and IL6ST (CD130/gp130) knockout samples were subjected to SDS-PAGE. ab283685 and ab181602 (Rabbit anti-GAPDH loading control) were incubated for 1 hour at room temperature at 1/1000 dilution and 1/200, 000 dilution respectively. Blots were developed with Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) secondary antibody at 1/50, 000 dilution and 1/100, 000 dilution for 1 hour at room temperature before imaging. The blot was developed on a BIO-RAD® ChemiDoc™ MP instrument using the ECL technique.
Samples are non-boiled as boiling may cause protein aggregates.
Exposure time: 70 seconds
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All lanes : Anti-CD130 (gp130) antibody [EPR21732] (ab217671) at 1/1000 dilution
Lane 1 : Wild-type A549 cell lysate
Lane 2 : IL6ST knockout A549 cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 104 kDa
Observed band size: 130 kDa why is the actual band size different from the predicted?Lanes 1-2: Merged signal (red and green). Green - ab217671 observed at 130 kDa. Red - loading control ab7291 observed at 50 kDa.
ab217671 Anti-CD130 (gp130) antibody [EPR21732] was shown to specifically react with CD130 (gp130) in wild-type A549 cells. Loss of signal was observed when knockout cell line ab266938 (knockout cell lysate ab257207) was used. Wild-type and CD130 (gp130) knockout samples were subjected to SDS-PAGE. ab217671 and Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) were incubated overnight at 4°C at 1 in 1000 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.
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Allele-1: 4 bp deletion in exon4
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Allele-2: 1 bp insertion in exon 4.
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Representative images of IL6ST knockout A549 cells, low and high confluency examples (top left and right respectively) and wild-type A549 cells, low and high confluency (bottom left and right respectively) showing typical adherent, epithelial-like morphology. Images were captured at 10X magnification using a EVOS XL Core microscope.
Protocols
Datasheets and documents
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SDS download
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Datasheet download
References (0)
ab266938 has not yet been referenced specifically in any publications.