Human RAF1 knockout HeLa cell line
- Advanced Validation
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- WB
Lab
Western blot - Human RAF1 knockout HeLa cell line (AB264978)
Lanes 1- 2 : Merged signal (red and green). Green - ab181115 observed at 73 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.
ab181115 was shown to react with Raf1 in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab264978 (knockout cell lysate ab257126) was used. Wild-type HeLa and Raf1 knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab181115 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 20000 dilution and a 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.
All lanes:
Western blot - Anti-Raf1 antibody [EP4969] - N-terminal (<a href='/en-us/products/primary-antibodies/raf1-antibody-ep4969-n-terminal-ab181115'>ab181115</a>) at 1/20000 dilution
Lane 1:
Wild-type HeLa cell lysate at 20 µg
Lane 2:
RAF1 knockout HeLa cell lysate at 20 µg
Lane 2:
Western blot - Human RAF1 knockout HeLa cell line (ab264978)
Predicted band size: 73 kDa
Observed band size: 73 kDa
false
- Sanger seq
Unknown
Sanger Sequencing - Human RAF1 knockout HeLa cell line (AB264978)
Homozygous : 2 bp insertion in exon 2.
Complementary Products
Primary Antibodies
AB181115
Anti-Raf1 antibody [EP4969] - N-terminal
KO Validated|RabMAb|Recombinant|Lab Essentials|20ul selling size
![Immunocytochemistry/ Immunofluorescence - Anti-Raf1 antibody [EP4969] - N-terminal (AB181115)](https://content.abcam.com/products/images/raf1-antibody-ep4969-n-terminal-ab181115--immunocytochemistry-immunofluorescence-img47165.jpg)
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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, 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.
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
DMEM (High Glucose) + 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
Raf1 regulates important cellular processes by activating downstream kinases in response to external stimuli. Raf1 forms a complex with other proteins such as Ras facilitating its role as an essential component of the MAPK/ERK pathway. Upon activation by Ras Raf1 phosphorylates and activates MEK1 and MEK2 which in turn activate the ERK1 and ERK2. This signaling axis is involved in controlling gene expression and cellular proliferation.
Pathways
Raf1 is integrally involved in the MAPK/ERK pathway which is critical for transducing signals from growth factors and mitogens. It relates closely with proteins such as Ras MEK and ERK in this pathway. The pathway is important for regulating cellular responses to various stimuli and is particularly involved in processes such as cell cycle control and apoptosis.
Quality control
STR analysis
CSF1PO, D13S317, D7S820, D5S818, TH01, D16S539, TPOX
Cell culture
Biosafety level
EU: 2 US: 2
Adherent/suspension
Adherent
Gender
Female
Target data
Product promise
Please note: All products are 'FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES'.
For licensing inquiries, please contact partnerships@abcam.com