Human GRB2 knockout HCT116 cell line (ab273715)
Overview
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Product name
Human GRB2 knockout HCT116 cell line -
Parental Cell Line
HCT116 -
Organism
Human -
Mutation description
Knockout achieved by using CRISPR/Cas9, Homozygous: 1 bp deletion in allele 1 and 2 bp deletion in allele 2 in exon 2 -
Passage number
<20 -
Knockout validation
Sanger Sequencing, Western Blot (WB) -
Tested applications
Suitable for: WBmore details -
Biosafety level
1 -
General notes
Recommended control: Human wild-type HCT116 cell line (ab273730). 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: McCoY5a + 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 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.
Properties
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Number of cells
1 x 106 cells/vial, 1 mL -
Viability
~80% -
Adherent /Suspension
Adherent -
Tissue
Colon -
Cell type
epithelial -
Disease
Carcinoma -
Gender
Male -
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
Adapter protein that provides a critical link between cell surface growth factor receptors and the Ras signaling pathway.
Isoform GRB3-3 does not bind to phosphorylated epidermal growth factor receptor (EGFR) but inhibits EGF-induced transactivation of a RAS-responsive element. Isoform GRB3-3 acts as a dominant negative protein over GRB2 and by suppressing proliferative signals, may trigger active programmed cell death. -
Sequence similarities
Belongs to the GRB2/sem-5/DRK family.
Contains 1 SH2 domain.
Contains 2 SH3 domains. -
Domain
The SH3 domains mediate interaction with RALGPS1 and SHB. -
Cellular localization
Golgi apparatus. - 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 ab273715 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: 25 kDa.
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Notes |
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WB
Use at an assay dependent concentration. Predicted molecular weight: 25 kDa. |
Images
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All lanes : Anti-GRB2 antibody [81/GRB2] (ab281846) at 1/1000 dilution
Lane 1 : Wild type HCT116 (human colorectal carcinoma epithelial cell), whole cell lysate
Lane 2 : GRB2 knockout HCT116 (ab273715) whole cell lysate
Lane 3 : HeLa (human epithelial cell line from cervix adenocarcinoma), whole cell lysate
Lane 4 : 293T ( human embryonic kidney epithelial cell), whole cell lysate
Lysates/proteins at 20 µg per lane.
Secondary
All lanes : Goat Anti-Mouse IgG H&L (IRDye® 800CW) (ab216772) and Goat Anti-Rabbit IgG H&L (IRDye® 680RD) (ab216777) at 1/10000 dilution
Predicted band size: 25 kDa
Observed band size: 26 kDa why is the actual band size different from the predicted?Blocking and diluting buffer and concentration: Intercept® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS
Lanes 1-4: Merged signal (red and green). Green - ab281846 observed at 26KDa. Red - loading control ab181602 (Rabbit monoclonal [EPR16891] to GAPDH) observed at 36 kDa.
Lanes 1-2: ab281846 Anti-GRB2 antibody was shown to react with GRB2 in HCT116 cells in Western blot. Loss of signal was observed when GRB2 knockout cell line ab273715 (knockout cell lysate ab275248) was used. Wild-type and GRB2 knockout samples were subjected to SDS-PAGE.
ab281846 and Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) were incubated at 4? overnight at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 680CW) preadsorbed (ab216777) and Goat anti-Mouse IgG H&L (IRDye® 800RD) preadsorbed (ab216772) secondary antibodies at 1 in 10000 dilution for 1 hour at room temperature before imaging. -
All lanes : Anti-GRB2 antibody [Y301] (ab32111) at 1/2000 dilution
Lane 1 : Wild-type HCT116 cell lysate
Lane 2 : GRB2 knockout HCT116 cell lysate
Lane 3 : HeLa cell lysate
Lane 4 : HEK293 cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 25 kDa
Observed band size: 25 kDaLanes 1 - 4: Merged signal (red and green). Green - ab32111 observed at 25 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.
ab32111 was shown to react with GRB2 in wild-type HCT 116 cells in western blot with loss of signal observed in GRB2 knockout cell line ab273715 (GRB2 knockout cell lysate ab275248). Wild-type and GRB2 knockout HCT 116 cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween®) before incubation with ab32111 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4°C at a 1 in 2000 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 hour at room temperature before imaging.
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Allele-1: 2 bp deletion in exon 2 .
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Allele-2: 1 bp deletion in exon 2 .
Protocols
Datasheets and documents
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SDS download
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Datasheet download
References (0)
ab273715 has not yet been referenced specifically in any publications.