Human GRB2 knockout HCT116 cell line
- Advanced Validation
- What is this?
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GRB2 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: 1 bp deletion in allele 1 and 2 bp deletion in allele 2 in exon 2. 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
ASH, Abundant SRC homology, Adapter protein GRB2, Ash protein, EGFRBP GRB2, Epidermal growth factor receptor binding protein, Epidermal growth factor receptor binding protein GRB2, GRB2 adapter protein, GRB2_HUMAN, Grb3 3, Growth factor receptor bound protein 3, Growth factor receptor-bound protein 2, HT027, MST084, MSTP084, NCKAP2, OTTHUMP00000166096, OTTHUMP00000166097, OTTHUMP00000166098, Protein ash, SEM5, SH2/SH3 adapter GRB2
- WB
Unknown
Western blot - Human GRB2 knockout HCT116 cell line (AB273715)
Lanes 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° 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.
All lanes:
Western blot - Anti-GRB2 antibody [Y301] (<a href='/en-us/products/primary-antibodies/grb2-antibody-y301-ab32111'>ab32111</a>) at 1/2000 dilution
Lane 1:
Wild-type HCT116 cell lysate at 20 µg
Lane 2:
GRB2 knockout HCT116 cell lysate at 20 µg
Lane 2:
Western blot - Human GRB2 knockout HCT116 cell line (ab273715)
Lane 3:
HeLa cell lysate at 20 µg
Lane 4:
HEK293 cell lysate at 20 µg
Predicted band size: 25 kDa
Observed band size: 25 kDa
false
- WB
Lab
Western blot - Human GRB2 knockout HCT116 cell line (AB273715)
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:
Western blot - Anti-GRB2 antibody [81/GRB2] (<a href='/en-us/products/primary-antibodies/grb2-antibody-81-grb2-ab281846'>ab281846</a>) at 1/1000 dilution
Lane 1:
Wild type HCT116 (human colorectal carcinoma epithelial cell), whole cell lysate at 20 µg
Lane 2:
GRB2 knockout HCT116 (ab273715) whole cell lysate at 20 µg
Lane 2:
Western blot - Human GRB2 knockout HCT116 cell line (ab273715)
Lane 3:
HeLa (human epithelial cell line from cervix adenocarcinoma), whole cell lysate at 20 µg
Lane 4:
293T (human embryonic kidney epithelial cell), whole cell lysate at 20 µg
Secondary
All lanes:
Goat Anti-Mouse IgG H&L (IRDye® 800CW) (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-h-l-irdye-800cw-preadsorbed-ab216772'>ab216772</a>) and Goat Anti-Rabbit IgG H&L (IRDye® 680RD) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-680rd-preadsorbed-ab216777'>ab216777</a>) at 1/10000 dilution
Predicted band size: 25 kDa
Observed band size: 26 kDa
false
- Sanger seq
Unknown
Sanger Sequencing - Human GRB2 knockout HCT116 cell line (AB273715)
Allele-1 : 2 bp deletion in exon 2 .
- Sanger seq
Unknown
Sanger Sequencing - Human GRB2 knockout HCT116 cell line (AB273715)
Allele-2 : 1 bp deletion in exon 2 .
Reactivity data
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.
What's included?
Properties and storage information
Gene name
Gene editing type
Gene editing method
Knockout validation
Zygosity
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.
Culture medium
McCoY5a + 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
GRB2 functions as an adaptor protein that connects activated receptor tyrosine kinases to intracellular pathways. It often forms complexes with other proteins such as the SOS protein which further propagates signaling cascades critical for cell proliferation and differentiation. GRB2's ability to mediate these interactions contributes to cellular responses to external stimuli including growth factors and hormones.
Pathways
GRB2 plays an important role in the Ras-MAPK signaling pathway. It interacts with proteins like SOS and Ras enabling signal transduction from the cell surface to the nucleus which is vital for processes such as cell growth and survival. GRB2 is also implicated in the PI3K-Akt pathway connecting it to another set of signaling proteins that regulate metabolism growth and survival.
Quality control
STR analysis
CSF1PO, D13S317, D7S820, D5S818, TH01, D16S539, TPOX
Cell culture
Biosafety level
EU: 1 US: 1
Adherent/suspension
Adherent
Gender
Male
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Product promise
Please note: All products are 'FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES'.
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