Product nameHuman BRAF knockout HeLa cell line
Parental Cell LineHeLa
Mutation descriptionKnockout achieved by using CRISPR/Cas9, 10 bp deletion in exon 3 and 1 bp insertion in exon 3
Knockout validationSanger Sequencing, Western Blot (WB)
Recommended control: Human Wild Type HeLa cell line (ab255928)
Please note a wild type cell line is not automatically included with a KO 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, supplemented with 10% (v/v) DMSO
Handling procedure: Upon arrival, the vial should be stored in liquid nitrogen vapor phase and not at -80°C. Storage at -80°C will result in loss of viability.
1. Thaw the vial in 37°C water bath approximately 1-2 minutes.
2. Transfer the cell suspension to a 15 mL conical tube with pre-warmed 5 mL complete medium DMEM+10% FBS, spin 125×g for approximately 5 minutes at room temperature.
3. Resuspend the cell pellet with 1 mL pre-warmed complete medium DMEM+10% FBS and dispense into a 25 cm2 culture flask containing 10 mL pre-warmed complete complete medium DMEM+10% FBS.
4. Incubate the culture at 37°C incubator with 5% CO2.
5. A subcultivation ratio of 1:4-1:6 is recommended. Cells should be passaged when cells grow splitting at 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.
Number of cells1 x 106 cells/vial, 1 mL
STR AnalysisAmelogenin 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
Antibiotic resistancePuromycin 1.00µg/ml
Storage instructionsShipped on Dry Ice. Store in liquid nitrogen.
Storage bufferConstituents: 8.7% DMSO, 2% Cellulose, methyl ether
FunctionInvolved in the transduction of mitogenic signals from the cell membrane to the nucleus. May play a role in the postsynaptic responses of hippocampal neuron.
Tissue specificityBrain and testis.
Involvement in diseaseNote=Defects in BRAF are found in a wide range of cancers.
Defects in BRAF may be a cause of colorectal cancer (CRC) [MIM:114500].
Defects in BRAF are involved in lung cancer (LNCR) [MIM:211980].
Defects in BRAF are involved in non-Hodgkin lymphoma (NHL) [MIM:605027]. NHL is a cancer that starts in cells of the lymph system, which is part of the body's immune system. NHLs can occur at any age and are often marked by enlarged lymph nodes, fever and weight loss.
Defects in BRAF are a cause of cardiofaciocutaneous syndrome (CFC syndrome) [MIM:115150]; also known as cardio-facio-cutaneous syndrome. CFC syndrome is characterized by a distinctive facial appearance, heart defects and mental retardation. Heart defects include pulmonic stenosis, atrial septal defects and hypertrophic cardiomyopathy. Some affected individuals present with ectodermal abnormalities such as sparse, friable hair, hyperkeratotic skin lesions and a generalized ichthyosis-like condition. Typical facial features are similar to Noonan syndrome. They include high forehead with bitemporal constriction, hypoplastic supraorbital ridges, downslanting palpebral fissures, a depressed nasal bridge, and posteriorly angulated ears with prominent helices. The inheritance of CFC syndrome is autosomal dominant.
Defects in BRAF are the cause of Noonan syndrome type 7 (NS7) [MIM:613706]. Noonan syndrome is a disorder characterized by facial dysmorphic features such as hypertelorism, a downward eyeslant and low-set posteriorly rotated ears. Other features can include short stature, a short neck with webbing or redundancy of skin, cardiac anomalies, deafness, motor delay and variable intellectual deficits.
Defects in BRAF are the cause of LEOPARD syndrome type 3 (LEOPARD3) [MIM:613707]. LEOPARD3 is a disorder characterized by lentigines, electrocardiographic conduction abnormalities, ocular hypertelorism, pulmonic stenosis, abnormalities of genitalia, retardation of growth, and sensorineural deafness.
Note=A chromosomal aberration involving BRAF is found in pilocytic astrocytomas. A tandem duplication of 2 Mb at 7q34 leads to the expression of a KIAA1549-BRAF fusion protein with a constitutive kinase activity and inducing cell transformation.
Sequence similaritiesBelongs to the protein kinase superfamily. TKL Ser/Thr protein kinase family. RAF subfamily.
Contains 1 phorbol-ester/DAG-type zinc finger.
Contains 1 protein kinase domain.
Contains 1 RBD (Ras-binding) domain.
Cellular localizationNucleus. Cytoplasm. Cell membrane. Colocalizes with RGS14 and RAF1 in both the cytoplasm and membranes.
- Information by UniProt
KO cell lysates
All lanes : Anti-BRAF antibody [EP152Y] (ab33899) at 1/1000 dilution
Lane 1 : HeLa WT
Lane 2 : BRAF HeLa KO
Lane 3 : HAP1 WT
Lane 4 : BRAF HAP1 KO
Lysates/proteins at 20 µg per lane.
All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/20000 dilution
Observed band size: 90 kDa why is the actual band size different from the predicted?
ab33899 Anti-BRAF antibody [EP152Y] was shown to specifically react with BRAF in HeLa wild type cells (ab255928). Loss of signal was observed when knockout cell line ab265373 (knockout cell lysate ab257078) was used. Wild type and BRAF knockout samples were subjected to SDS-PAGE. ab33899 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 dilution 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.
Allel-1: 10 bp deletion in exon 3.
Allel-2: 1 bp insertion in exon 3.
To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.
ab265373 has not yet been referenced specifically in any publications.